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WO2011049964A1 - Détection et quantification d'arn - Google Patents

Détection et quantification d'arn Download PDF

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Publication number
WO2011049964A1
WO2011049964A1 PCT/US2010/053224 US2010053224W WO2011049964A1 WO 2011049964 A1 WO2011049964 A1 WO 2011049964A1 US 2010053224 W US2010053224 W US 2010053224W WO 2011049964 A1 WO2011049964 A1 WO 2011049964A1
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WO
WIPO (PCT)
Prior art keywords
rna
rna molecule
dna
template
specific
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PCT/US2010/053224
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English (en)
Inventor
Zhen Huang
Sarah M. Spencer
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Lab Scientific Group
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Publication of WO2011049964A1 publication Critical patent/WO2011049964A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to methods and devices for detecting nucleic acid sequences, the presence of which is a positive indicator of a pathogenic agent, contaminant, and/or normal or abnormal genes or gene products.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q.beta. replicase amplification Q.beta.RA
  • NASBA nucleic acid sequence based amplification
  • 3SR self-sustained replication
  • the preparation of the target nucleic acid is a procedural impediment required for subsequent steps such as amplification and detection.
  • Target nucleic acid preparation is time and labor intensive and, thus, generally unsuitable for a clinical setting, where rapid and accurate results are required.
  • Another problem, which is particularly pronounced when using PCR and SDA, is the necessity for empirically determining optimal conditions for target nucleic acid amplification for each target.
  • conditions required for standardizing quantitation assessments can also vary from sample to sample. This lack of precision manifests itself most dramatically when the diagnostic assay is implemented in multiplex format, that is, in a format designed for the simultaneous detection of several different target sequences.
  • compositions, articles, and methods aredirected to identification of nucleic acids, such as RNA molecules.
  • methods for detecting at least one specific RNA molecule in a population comprising a plurality of different RNA molecules comprising: a method for detecting at least one specific RNA molecule in a population comprising a plurality of different RNA molecules, said method comprising: making a hybrid template comprising a first portion and a second portion, wherein said first and second portion of said hybrid template are operably linked, and wherein the first portion is an RNA sequence complementary to an internal sequence of said specific RNA molecule and said second portion is a DNA sequence complementary to a region proximal to the internal sequence of said specific RNA molecule;
  • the method of the invention is directed to the detection of a specific messenger RNA (mRNA) molecule.
  • mRNA messenger RNA
  • RNA molecules comprising a plurality of different RNA molecules is derived from a sample.
  • the sample is a biological sample.
  • detecting a specific RNA molecule is a positive indicator of a presence of a microorganism, pathogen, or gene in a sample.
  • the DNA and/or RNA sequences of the hybrid template are modified.
  • Exemplary modifications of the DNA sequences of a hybrid template of the method include, but are not limited to, 3' amino group modification.
  • Exemplary modifications of the RNA sequences of a hybrid template of the method include, but are not limited to, 2'-0-methyl group modification.
  • the riboendonuclease used is RNase H.
  • the extension can be performed by a polymerase.
  • the polymerase is a Klenow DNA polymerase.
  • RNA chip a hybrid template bound solid matrix
  • RNA chips may comprise a plurality of different hybrid templates.
  • an RNA chip may comprise a plurality of different hybrid templates that are specific for a single microorganism, pathogen, or gene.
  • Exemplary hybrid templates include, but are not limited to, SEQ ID NOs: 14, 15, 5, 6, 20, 23, 26, and 29. See, for example, FIGS.
  • an RNA chip may comprise a plurality of different hybrid templates that are specific for a plurality of microorganisms, pathogens, or genes.
  • a hybrid template bound solid matrix of the invention such as an RNA chip
  • Also disclosed are methods for detecting at least one specific RNA molecule in a population comprising a plurality of different RNA molecules comprising: (a) making a hybrid template comprising a middle portion and two portions flanking the middle portion, wherein said middle and flanking portions of said hybrid template are operably linked, and wherein the middle portion comprises an RNA sequence complementary to an internal sequence of said specific RNA molecule and said flanking portions comprise DNA sequences complementary to regions flanking the internal sequence of said specific RNA molecule; (b) binding said hybrid template to said specific RNA molecule, wherein said binding produces a complex comprising said specific RNA molecule and said hybrid template and said binding results in formation of a double stranded RNA/RNA duplex at the internal sequence of said specific RNA molecule and double stranded RNA/DNA duplexes at the regions flanking the internal sequence of said specific RNA molecule; (c) digesting said complex with a riboendonuclease capable of digesting double- stranded
  • a hybrid template comprising a middle (or central) and flanking portions (i.e., a tripartite hybrid template) is used, the method is directed to the detection of a specific messenger RNA (mRNA) molecule.
  • mRNA messenger RNA
  • the population comprising a plurality of different RNA molecules is derived from a sample.
  • the sample is a biological sample.
  • detecting a specific RNA molecule is a positive indicator of a presence of a microorganism, pathogen, or gene in a sample.
  • the DNA and/or RNA sequences of the tripartite hybrid template are modified.
  • Exemplary modifications of the DNA sequences of a tripartite hybrid template of the method include, but are not limited to, 3' amino group modification.
  • Exemplary modifications of the RNA sequences of a hybrid template of the method include, but are not limited to, 2'-0-methyl group modification.
  • the riboendonuclease used is RNase H.
  • the extension can be performed by a polymerase.
  • the polymerase is a Klenow DNA polymerase.
  • a tripartite hybrid template is bound to a solid matrix to produce a hybrid template bound solid matrix.
  • hybrid template bound solid matrices e.g., an RNA chip
  • a tripartite hybrid template(s) is bound to a solid matrix
  • RNA chips may comprise a plurality of different tripartite hybrid templates.
  • an RNA chip may comprise a plurality of different tripartite hybrid templates that are specific for a single microorganism, pathogen, or gene.
  • an RNA chip may comprise a plurality of different tripartite hybrid templates that are specific for a plurality of microorganisms, pathogens, or genes.
  • RNA chip for detecting a specific RNA molecule in a sample
  • the RNA chip comprises different bound tripartite hybrid templates, and detecting a specific RNA molecule in a sample is a positive indicator of a presence of a microorganism, pathogen, or gene in the sample.
  • buffer solutions for the performance of the hybridization, digestion and extension steps of the disclosed methods, the buffer solution comprising a digestion buffer, a hybridization buffer, and an extension buffer.
  • the present invention also encompasses a kit.
  • a kit comprises materials for practicing the methods described herein, including: RNase H; Klenow DNA polymerase; a buffer compatible with RNase H and Klenow DNA polymerase activities; a positive control RNA; a hybrid template and/or tripartite hybrid template specific for said control RNA; and instructional materials.
  • FIG. 1 shows an autoradiogram and procedural flowchart depicting labeling of RNA at an internal site after RNase H digestion.
  • Lane 1 Klenow extension of RNA50 without the RNase H digestion; lane 2, digestion and extension on a control template (DNA20. 10); lane 3, Klenow extension of RNA50 on the DNA template (DNA20.8) after the RNase H digestion on the DNA-2'-O-Me-RNA20.8 hybrid template; lane 4, Klenow extension of RNA50 on the same DNA-2'-O-Me-RNA20.8 hybrid template after RNase H digestion.
  • the bold sequences are sequences for RNase H digestion guidance and Klenow extension template, and the underlined sequences are complementary to the RNA substrate after the RNase H digestion.
  • RNA50 SEQ ID NO: 7
  • Hybrid Template DNA-2'-O-Me-RNA20.8 SEQ ID NO: 3
  • Digested RNA40 SEQ ID NO: 10
  • Template bound to RNA40 SEQ ID NO: 11
  • Labeled RNA41 SEQ ID NO: 12
  • Template bound to RNA41 SEQ ID NO: 11
  • FIG. 2 shows an autoradiogram and cartoon illustrating selective labeling and detection of lacZ mRNA in E. coli total RNA via RNase H digestion and DNA polymerase extension.
  • Lane 1 marker; lane 2, total RNA (0.4 ⁇ g) isolated from IPTG induced E. coli cells; lane 3, total RNA (0.4 ⁇ g) isolated from glucose repressed E. coli cells; lane 4, IPTG- induced total RNA (0.4 g), no Klenow.
  • the autoradiography film was exposed for one day before development.
  • FIG. 3 shows an autoradiogram and schematic depicting the labeling and detection of RNA31 and lacZ mRNA on template DNA-2'-0-Me-RNA35.1. 1 ⁇ .
  • RNA31 l0.sup.-18 moles of RNA31 ; lane 8, IPTG-induced E. coli total RNA (1 ⁇ g); lane 9, glucose-repressed E. coli total RNA (1 ⁇ g); lane 10, yeast total mRNA (10 ng) isolated from lacZ mRNA-expressing yeast system.
  • FIG. 4 shows a cartoon illustrating the detection of mRNA with enzyme labeling and chemiluminescence.
  • FIG. 5 shows an autoradiogram which visualizes selective labeling of lacZ mRNA on a 96-well plate.
  • Spot 1 negative control
  • Spot 2 total mRNA isolated from the glucose culture
  • Spot 3 total mRNA isolated from the galactose culture
  • Spot 4 positive control
  • RNA24 RNA24 addition to experiment in Spot 2
  • FIG. 6 is a flowchart of RNA specific detection on a plate or microchip.
  • FIG. 7 is a stick figure illustrating the general design of a hybrid template.
  • FIG. 8 shows a nucleic acid sequence of a bacterial Rps F gene (SEQ ID NO: 13). Nucleic acid sequences comprising Template 1 (SEQ ID NO: 14) and Template 2 (SEQ ID NO: 15) and their targeting sequences (SEQ ID Nos: 16 and 17, respectively) in the Rps F gene are also indicated.
  • FIGS. 9 A and 9B show a nucleic acid sequence of an E. coli lacZ gene open reading frame encoding beta-galactosidase (EC 3.2.1.23) (SEQ ID NO: 18). Nucleic acid sequences comprising Template 1 (SEQ ID NO: 5) and Template 2 (SEQ ID NO: 6) and their targeting sequences in the E. coli lacZ gene open reading frame are also indicated.
  • FIG. 10 shows a nucleic acid sequence of an exoA gene of S. meliloti strain 1021 (SEQ ID NO: 19). Nucleic acid sequences comprising a Template (SEQ ID NO: 20) and its targeting sequences (SEQ ID NO: 21) in the exoA gene are also indicated.
  • FIG. 11 shows a nucleic acid sequence of a PF2NC15 polyprotein gene of
  • FIG. 12 shows a nucleic acid sequence of a human immunodeficiency virus- 1 (HIV-1) envelope (env) gene (SEQ ID NO: 25). Nucleic acid sequences comprising a Template (SEQ ID NO: 26) and its targeting sequences (SEQ ID NO: 27) in the env gene are also indicated.
  • HSV-1 human immunodeficiency virus- 1 envelope (env) gene
  • FIG. 13 shows a nucleic acid sequence of a SARS gene (SEQ ID NO: 28).
  • Nucleic acid sequences comprising a Template (SEQ ID NO: 29) and its targeting sequences (SEQ ID NO: 30) in the SARS gene are also indicated.
  • FIG. 14 shows a schematic flow chart of specific RNA detection on a microplate.
  • FIGS. 15A and B show autoradiograms which visualize enzymatic detection of RNA on 96-well microplates.
  • Target RNA24.1 (1 pmole) and template DNA35.1 (100 pmole).
  • B Detection sensitivity studies were as follows: well 1, no RNA24.1 ; well 2, 1 x 10 "15 mole; well 3, 1. times.10. sup. -4 mole; well 4, 1. times. lO.sup.- 13 mole.
  • the film was exposed for one hour (A) or five hours (B) after substrate addition.
  • FIG. 16 shows an autoradiogram revealing selective detection of lacZ mRNA on a microplate.
  • Total mRNA and RNA24.1 used for each experiment were 0.1 ⁇ g and 10 fmole, respectively (6 hr exposure).
  • Well 1 galactose-induced mRNA; well 2, glucose-repressed mRNA; well 3, no RNA (negative control); well 4, glucose-repressed mRNA and RNA24.1 ; and well 5, RNA24.1 (positive control).
  • Figure 17 A and 17B show hybridization kinetics and rapid RNA detection.
  • Figure 18 shows RNA detection on RNA microchip with single-nucletide discrimination via the RNase H digestion and Klenow extension at 60° C.
  • Figure 19 shows the detection sensitivity and selective detection of an individual mRNA.
  • 19A shows the RNA detection sensitivity; Image 1-5: 0, 1, 10, 25 and 50 fmole RNA respectively.
  • 19B shows the detection of lacL mRNA in the IPTG-induced total RNA; Image 1-4: no RNA, the glucose-suppressed total RNA, a mixture of the glucose- suppressed total RNA and the lacL mRNA synthetic fragment, and the IPTG-induced total RNA, respectively.
  • Figure 20 shows the scheme of rapid RNA detection using enzymatic reactions on RNA chips
  • Figure 21 shows the activation of the silicon or glass microchip surface.
  • Figure 22A, B, C, D, E, and F show simultaneous and selective detection of multi-pathogen RNAs on RNA microchip.
  • the RNA microchip was immobilized with the corresponding detecting probes (2x2 spots for each probe) for the lacZ mRNA (lacZ), Bacillus anthracis RNA (BA), bird flu RNA (BF), and swine flu RNA (SF).
  • lacZ lacZ
  • BA Bacillus anthracis RNA
  • BF bird flu RNA
  • SF swine flu RNA
  • A) The SF RNA and its detecting probe are shown here as examples; the other RNA sequences and their probes are presented in the supporting information.
  • SF RNA was specifically detected by incorporating the biotin-labeled dGTP and dATP into the RNA.
  • Consisting essentially of in embodiments refers, for example, to a surface composition, a method of making or using a surface composition, formulation, or composition on the surface of the biosensor, and articles, devices, or apparatus of the disclosure, and can include the components or steps listed in the claim, plus other components or steps that do not materially affect the basic and novel properties of the compositions, articles, apparatus, and methods of making and use of the disclosure, such as particular reactants, particular additives or ingredients, a particular agents, a particular cell or cell line, a particular surface modifier or condition, a particular ligand candidate, or like structure, material, or process variable selected.
  • Items that may materially affect the basic properties of the components or steps of the disclosure or may impart undesirable characteristics to the present disclosure include, for example, decreased affinity of the cell for the biosensor surface, aberrant affinity of a stimulus for a cell surface receptor or for an intracellular receptor, anomalous or contrary cell activity in response to a ligand candidate or like stimulus, and like characteristics.
  • compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these molecules may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning
  • any subset or combination of these is also disclosed.
  • the subgroup of A-E, B-F, and C-E would be considered disclosed.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
  • a "detection label” or like terms refers to any molecule or moiety which can be detected by, such as flourescence, radioactivity, phosphorescence, or the like.
  • extended RNA molecule refers to an RNA molecule which has been extended or made longer.
  • extended RNA molecules can comprise at least one detectable label.
  • the extended RNA molecule can be shorter than the original specific RNA molecule that binds to a hybrid template but will be longer than the RNase H treated RNA molecule.
  • hybrid template refers to the combination of one or more different things on one template.
  • a hybrid template can comprise a nucleic acid template comprising both DNA and RNA.
  • the hybrid template comprises two DNA portions and one RNA portion.
  • the hybrid template comprises one DNA portion and one RNA portion. 11.
  • microorganism or "pathogen” refers to a variety of organisms. Microorganisms can include bacteria, fungi, archaea, and protists; microscopic plants (green algae); and animals such as plankton and the planarian. In some instances, viruses can be considered microorganisms. Pathogens are biological agents that can cause disease. Many microorganisms are also pathogens.
  • the terms "linked”, “operably linked” and “operably bound” and variants thereof mean, for purposes of the specification and claims, to refer to fusion, bond, adherence or association of sufficient stability to withstand conditions encountered in single molecule applications and/or the methods and systems disclosed herein, between a combination of different molecules such as, but not limited to: between a detectable label and nucleotide, between a detectable label and a linker, between a nucleotide and a linker, between a protein and a functionalized nanocrystal; between a linker and a protein; and the like.
  • the label in a labeled polymerase, is operably linked to the polymerase in such a way that the resultant labeled polymerase can readily participate in a polymerization reaction.
  • operable linkage or binding may comprise any sort of fusion, bond, adherence or association, including, but not limited to, covalent, ionic, hydrogen, hydrophilic, hydrophobic or affinity bonding, affinity bonding, van der Waals forces, mechanical bonding, etc.
  • the term "positive indicator” refers to anything that provides the ability to positively identify a specific sample.
  • a detectable label on an extended RNA molecule can act as a positive indicator by allowing one to positively identify that particular extended RNA molecule as the specific RNA molecule of interest.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • riboendonuclease refers to an enzyme that can cleave RNA within the RNA molecule and does not require a free 3' or 5' terminus. They can be specific or nonspecific.
  • RNase H which cleaves RNA found in an RNA/DNA duplex.
  • sample or like terms is meant an animal, a plant, a fungus, etc.; a natural product, a natural product extract, etc.; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
  • Solid matrix for use in the disclosed method can include any solid material to which components of the assay can be adhered or coupled.
  • substrates include, but are not limited to, materials such as acrylamide, cellulose, nitrocellulose, glass, silicon chip, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids.
  • Substrates can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, woven fibers, shaped polymers, particles and microparticles.
  • Preferred forms of substrates are glass slides, plates and beads.
  • the most preferred form of beads are magnetic beads.
  • compositions, apparatus, and methods of the disclosure include those having any value or any combination of the values, specific values, more specific values, and preferred values described herein.
  • the disclosed methods, compositions, articles, and machines can be combined in a manner to comprise, consist of, or consist essentially of, the various components, steps, molecules, and composition, and the like, discussed herein.
  • RNA chip made using the described methods. Details pertaining to the method and products generated using the method are clearly set forth herein below.
  • Ribonuclease H is an endoribonuclease which specifically hydrolyzes the phosphodiester bonds of RNA which is hybridized to DNA. This enzyme does not digest single stranded nucleic acids, double- stranded DNA, or double stranded RNA.
  • Microarray analysis and real-time PCR are the most popular technologies in this area (Golub et al. (1999) Science 286:531-537; Trottier et al. (2002) J. Virol. Methods 103:89-99).
  • Microarrays comprised of oligonucleotides or complementary DNA (cDNA) have been used successfully in gene expression profiling studies. Such studies provide information on expression levels of individual genes and reveal patterns of coordinated gene expression. This information can be used in drug discovery, cancer monitoring, cancer type classification, and identification of microorganisms, viruses, and other pathogens in a sample (Golub, et al. 1999, supra; Young et al. (2002) J Virol Methods 103:27-39).
  • technologies directed to the use of high-density microarrays allow gene expression profiling of over tens of thousands of genes (Lockhart and Winzeler, 2000, supra).
  • oligonucleotide microarray procedure generally consists of the following steps: reverse transcription, DNA polymerization, transcription, biotin- strep tavidin interactions or antibody binding (or the like), and fluorophore labeling.
  • the signals are amplified during the transcription step and subsequent steps, wherein fluorescent labels are incorporated.
  • the fluorophores are activated by laser excitation to emit detectable fluorescent signals.
  • RNAs include, but are not limited to, viral and bacterial RNAs in RNA samples, or specific mRNA transcripts in samples comprising total RNA.
  • modified terminal RNA labeling methods which directly label and detect specific RNA molecules in a mixture. The methods are can fundamentally differ from existing methods that have been used to determine gene expression patterns in that it does not require reverse transcription, PCR, in vitro
  • RNA detection microchip technology is simple, rapid, accurate, sensitive, high- throughput, and cost-effective, it is an ideal assay for point-of-care disease diagnosis, detection of microbial contamination in food and/or water supplies, and pathogen detection in biodefense.
  • the above mentioned novel chip technology uses direct labeling/detection of a specific mRNA in total mRNA or a sample comprising total RNA.
  • a method wasevised to remove the 3 '-region which is conserved among most eukaryotic mRNA transcripts [e.g., the poly(A) tail and 3 '-untranslated region )3'-UTR)] from a specific mRNA and, thereby expose intrinsic 3'-sequences for labeling and detection.
  • the method can involve an Rnase H digestion protocol which takes advantage of the ability of Rnase H to digest RNA which has formed a duplex with a DNA sequence (Nakamura and Oda. (1991) Proc. Natl. Acad. Sci. USA 88:11535-11539).
  • the method can rely on the selection of a 2'-0-Me-RNA/DNA hybrid which binds to a specific mRNA and protects a unique internal sequence of the mRNA from Rnase H mediated digestion (via RNA/RNA duplex formation), but also binds/positions other regions of the mRNA (such as the 3 '-region), so as to render these regions susceptible to Rnase H digestion (via RNA/DNA duplex formation).
  • the overhang formed following Rnase H mediated digestion serves as a recognition/extension site for a DNA polymerase (e.g., Klenow) on the fragment of the specific mRNA whereby nucleotide labels may be incorporated to effect detection of the specific mRNA.
  • a DNA polymerase e.g., Klenow
  • the methods are based in part on a method developed by Huang and Szostak [(1996) Nucleic Acids Res. 24, 4360-1] for labeling the 3'-termini of RNA.
  • This method took advantage of a natural function of DNA polymerases: elongation of RNA primers on DNA templates. This observation was subsequently investigated further and shown to be applicable to the development of a method for labeling and detecting specific RNA transcripts.
  • Huang and Szostak [(2003) Anal Biochem 315: 129-133] discovered that the ready availability of short synthetic DNA template allows an RNA of known 3 '-terminal sequence to be selectively extended in a template-dependent manner at its 3 '-end, which facilitates labeling and detection of the specific RNA in an RNA mixture, without separation, purification, reverse transcription, or PCR.
  • the contents of each of Huang and Szostak [(1996) Nucleic Acids Res. 24, 4360-1] and Huang and Szostak [(2003) Anal Biochem 315: 129-133] are incorporated herein by reference in their entirety. Methodology relating to labeling and modification of RNA 3'-termini are also described in U.S. Pat. No. 6,238,865 (issued to Huang and Szostak), the entire contents of which is incorporated herein by reference.
  • the step of radioactive labeling method of Huang and Szostak [(2003), supra)] can also be modified to become an enzyme labeling method, which uses enzymes such as peroxidase or alkaline phosphatase to catalyze chemiluminescent reactions (Pollard-Knight et al. (1990) Anal. Biochem. 185, 84-89; Reddy et al. (1999) Biotechniques 26710-714). Details pertaining to using the method with various enzyme labeling method with various enzyme labeling method, which uses enzymes such as peroxidase or alkaline phosphatase to catalyze chemiluminescent reactions (Pollard-Knight et al. (1990) Anal. Biochem. 185, 84-89; Reddy et al. (1999) Biotechniques 26710-714). Details pertaining to using the method with various enzyme labeling method, which uses enzymes such as peroxidase or alkaline phosphatase to catalyze
  • RNAs can be labeled initially with antigens and subsequently labeled with enzymes, such as alkaline phosphatase (AP), which can catalyze a
  • chemiluminescent reaction Unlike fluorescence detection, wherein detection sensitivity is relatively low and laser excitation is required to generate fluorescent signals,
  • chemiluminescence detection sensitivity is high and excitation is not needed. These features can dramatically reduce the instrumental costs associated with detection. Moreover, an instrument which is not required to have laser excitation capabilities would also tend to be smaller and lighter than those used for fluorescence detection. Such aspects of the invention are well suited to the challenges associated with field detection and point-of-care.
  • a variety of DNA and RNA polymerases have been screened and examined for the ability to catalyze RNA 3 '-extension on a DNA template.
  • Enzymes including E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467), T4 DNA polymerase, T7 DNA polymerase, T7 RNA polymerase, M-MuLV reverse transcriptase, and Taq DNA polymerase have been tested for utility in the present methods by incubating each enzyme with a 5'-.sup.32P- labeled RNA, dNTPs, and a DNA template.
  • the Klenow fragment may be considered a preferred enzyme for use in 3 '-labeling reactions as described herein. See Huang and Szostak [(2003) Anal Biochem 315:129-133].
  • RNA terminal-labeling Three methods for RNA terminal-labeling are commonly used: 5 '-labeling with T4 polynucleotide kinase and [.gamma.-. sup.32P]-ATP (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor); 3'-labeling with T4 RNA ligase, and 3',5'-[5'-.sup.32P]-pCp (England and Uhlenbeck.
  • Specific labeling may be necessitated when analyzing, for example, a specific viral RNA, ribosomal RNA, or cellular mRNA in a total RNA sample.
  • separation steps are required to isolate the specific RNA from the mixture.
  • RNA of interest such as an mRNA, or other functional RNA
  • protein targets for analysis RNA of interest (such as an mRNA, or other functional RNA) or protein targets for analysis.
  • RNA sequence (6-80 nt.) intended for incorporation into a template should be analyzed using computer-assisted folding programs, such as Mfold (M. Zuker,
  • RNA sequence is predicted to form a secondary structure, a different RNA sequence should be considered.
  • Design (I) comprises a 5'-end DNA (1-30 nt.) and a 3'-end 2'-MeO-RNA (5-79 nt.).
  • the DNA is designated herein template DNA and the 2'-MeO-RNA is designated herein binding MeO-RNA.
  • the targeted RNA sequence section bound to the template DNA is referred to as the labeling region and the targeted RNA sequence section bound to the binding MeO-RNA sequence is referred to as the binding region.
  • Design (II) comprises 5'-end and 3'-end DNA sequences (1-30 nt. each) flanking the central 2'-MeO-RNA sequence (4-78 nt.).
  • the 5'-end DNA is called template DNA
  • the 3'- end DNA is called digestion DNA
  • the 2 '-MeO-RNA is called binding MeO-RNA. See FIG. 7.
  • the targeted RNA sequence section bound to the template DNA is referred to as the labeling region
  • the targeted RNA sequence section bound to the digestion DNA is referred to as the digestion region
  • the targeted RNA sequence section bound to the binding MeO- RNA sequence is referred to as the binding region.
  • the first several 5'- nucleotides (at least two nucleotides) in the labeling region may be selected to be a single kind of nucleotide, so as to produce, for example, a stretch of 5'-AAAA.
  • a hybrid template (I or II) may be chemically synthesized on solid phase and purified by HPLC or gel electrophoresis. Techniques directed to the synthesis and purification of such sequences are known in the art and routinely practiced.
  • FIGS. 8-13 provide nucleic acid sequences of a subset of exemplary genes, some of which are associated with various microorganisms and/or pathogens, which may be used in the detection methods of the present invention. Also presented in FIGS. 8-13 are sequences of hybrid templates useful in the method of the invention for detection of the specific gene (i.e., the RNA) indicated.
  • the specific gene i.e., the RNA
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • the conditions of hybridization to achieve selective hybridization can involve hybridization in high ionic strength solution (6.times.SSC or 6.times.SSPE) at a temperature that is about 12-25° C. below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5. degree. C. to 20° C. below the Tm.
  • hybridizations The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids).
  • DNA:DNA hybridization can be at about 68° C. (in aqueous solution) in 6.times.SSC or 6.times.SSPE followed by washing at 68° C.
  • Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • selective hybridization conditions are by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid.
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
  • This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k.sub.d, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k ⁇
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
  • Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
  • Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
  • homology it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions can provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.
  • composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.
  • detectable agents are useful in the disclosed methods.
  • the term "detectable label” refers to any molecule which can be detected.
  • Useful detectable labels include compounds and molecules that can be administered in vivo and subsequently detected.
  • Detectable labels useful in the disclosed compositions and methods include yet are not limited to radiolabels and fluorescent molecules.
  • the detectable label can be, for example, any moiety or molecule that facilitates detection, either directly or indirectly, preferably by a non-invasive and/or in vivo visualization technique.
  • a detectable label can be detectable by any known imaging techniques, including, for example, a radiological technique, a magnetic resonance technique, or an ultrasound technique.
  • Detectable labels can include, for example, a contrast agent.
  • the contrast agent can be, for example, Feridex.
  • the contrasting agent can be, for example, ionic or non-ionic.
  • the detectable label comprises a tantalum compound and/or a barium compound, e.g., barium sulfate.
  • the detectable label comprises iodine, such as radioactive iodine.
  • the detectable label comprises an organic iodo acid, such as iodo carboxylic acid, triiodophenol, iodoform, and/or tetraiodoethylene.
  • the detectable label comprises a non-radioactive detectable agent, e.g., a non-radioactive isotope.
  • a non-radioactive detectable agent e.g., iron oxide and Gd can be used as a non-radioactive detectable label in certain embodiments.
  • Detectable labels can also include radioactive isotopes, enzymes, fluorophores, and quantum dots (Qdot®).
  • the detection label can be an enzyme, biotin, metal, or epitope tag.
  • Other known or newly discovered detectable labels are contemplated for use with the provided compositions.
  • the detectable label comprises a barium compound, e.g., barium sulfate.
  • detectable labels include molecules which emit or can be caused to emit detectable radiation (e.g., fluorescence excitation, radioactive decay, spin resonance excitation, etc.), molecules which affect local electromagnetic fields (e.g., magnetic, ferromagnetic, ferromagnetic, paramagnetic, and/or superparamagnetic species), molecules which absorb or scatter radiation energy (e.g., chromophores and/or fluorophores), quantum dots, heavy elements and/or compounds thereof. See, e.g., detectable agents described in U.S. Publication No. 2004/0009122.
  • detectable labels include a proton-emitting molecules, a radiopaque molecules, and/or a radioactive molecules, such as a radionuclide like Tc-99m and/or Xe-13. Such molecules can be used as a
  • the disclosed compositions can comprise one or more different types of detectable labels, including any combination of the detectable labels disclosed herein.
  • Useful fluorescent labels include fluorescein isothiocyanate (FITC), 5,6- carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY ® , Cascade Blue ® , Oregon Green ® , pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dyeTM, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • FITC fluorescein isothiocyanate
  • Texas red nitrobenz-2-oxa-l,3-diazol-4
  • Examples of other specific fluorescent labels include 3- Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9
  • Dimethyl Amino Naphaline 5 Sulphonic Acid Dimethyl Amino Naphaline 5 Sulphonic Acid
  • Dansa Diamino Naphtyl Sulphonic Acid
  • Dansyl NH-CH3 Diamino Phenyl Oxydiazole
  • DAO Diamino Phenyl Oxydiazole
  • Dimethylamino-5-Sulphonic acid Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3,
  • Particularly useful fluorescent labels include fluorescein (5-carboxyfluorescein- N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • fluorescein dyes include 6- carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4- hexachlorofluorescein (HEX), 2',7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE), 2'- chloro-5'-fluoro-7',8'-fused phenyl- l,4-dichloro-6-carboxyfluorescein (NED), and 2'-chloro- 7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC).
  • Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ;
  • radioactive detectable labels include gamma emitters, e.g., the gamma emitters In-I ll, 1-125 and 1-131, Rhenium-186 and 188, and Br-77 (see. e.g., Thakur, M. L. et al., Throm Res. Vol. 9 pg. 345 (1976); Powers et al., Neurology Vol. 32 pg. 938 (1982); and U.S. Pat. No.
  • radioactive detectable labels can include, for example tritium, C-14 and/or thallium, as well as Rh-105, 1-123, Nd-147, Pm-151, Sm-153, Gd-159, Tb-161, Er-171 and/or Tl-201.
  • Tc-99m Technitium-99m
  • Tc- 99m is a gamma emitter with single photon energy of 140 keV and a half-life of about 6 hours, and can readily be obtained from a Mo-99/Tc-99 generator.
  • compositions comprising a radioactive detectable label can be prepared by coupling radioisotopes suitable for detection to the disclosed components and compositions. Coupling can be, for example, via a chelating agent such as
  • DTP A diethylenetriaminepentaacetic acid
  • DTA 4,7,10-tetraazacyclododecane-N- ,N',N",N"'- tetraacetic acid
  • metallothionein any of which can be covalently attached to the disclosed components, compounds, and compositions.
  • an aqueous mixture of technetium-99m, a reducing agent, and a water-soluble ligand can be prepared and then allowed to react with a disclosed component, compound, or composition.
  • Such methods are known in the art, see e.g., International Publication No. WO 99/64446.
  • compositions comprising radioactive iodine can be prepared using an exchange reaction.
  • a radio-iodine labeled compound can be prepared from the corresponding bromo compound via a tributylstannyl intermediate.
  • Magnetic detectable labels include paramagnetic contrasting agents, e.g., gadolinium diethylenetriaminepentaacetic acid, e.g., used with magnetic resonance imaging (MRI) (see, e.g., De Roos, A. et al., Int. J. Card. Imaging Vol. 7 pg. 133 (1991)).
  • Some preferred embodiments use as the detectable label paramagnetic atoms that are divalent or trivalent ions of elements with an atomic number 21, 22, 23, 24, 25, 26, 27, 28, 29, 42, 44, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.
  • Suitable ions include, but are not limited to, chromium(III), manganese(II), iron(II), iron(III), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) and ytterbium(III), as well as gadolinium(III), terbiurn(III), dysoprosium(III), holmium(III), and erbium(III).
  • Some preferred embodiments use atoms with strong magnetic moments, e.g., gadolinium(III).
  • compositions comprising magnetic detectable labels can be prepared by coupling the disclosed components, compounds, and compositions with a paramagnetic atom.
  • a paramagnetic atom for example, the metal oxide or a metal salt, such as a nitrate, chloride or sulfate salt, of a suitable paramagnetic atom can be dissolved or suspended in a water/alcohol medium, such as methyl, ethyl, and/or isopropyl alcohol.
  • the mixture can be added to a solution of an equimolar amount of the disclosed components, compounds, and compositions in a similar water/alcohol medium and stirred. The mixture can be heated moderately until the reaction is complete or nearly complete.
  • Insoluble compositions formed can be obtained by filtering, while soluble compositions can be obtained by evaporating the solvent. If acid groups on the chelating moieties remain in the disclosed compositions, inorganic bases (e.g., hydroxides, carbonates and/or bicarbonates of sodium, potassium and/or lithium), organic bases, and/or basic amino acids can be used to neutralize acidic groups, e.g., to facilitate isolation or purification of the composition.
  • inorganic bases e.g., hydroxides, carbonates and/or bicarbonates of sodium, potassium and/or lithium
  • organic bases e.g., organic bases, and/or basic amino acids
  • the detectable label can be coupled to the composition in such a way so as not to interfere with the ability of the compound to interact with the template.
  • the detectable label can be chemically bound to the compound.
  • the detectable label can be chemically bound to a moiety that is itself chemically bound to the compound, indirectly linking the imaging and the disclosed components, compounds, and compositions.
  • Example I Detection of a Specific mRNA in a Sample Comprised of Total RNA 1. Materials and Methods
  • DNA20.8 (5 '-TGAATCAGCATCTAGCTACG-3 ') (SEQ ID NO: 1), DNA20.10 (5'-GGCTACAGGAAG-GCCAGACG-3') (SEQ ID NO: 2), DNA-2'-O-Me-RNA20.8 [a hybrid template, 5'-d(TGAAT)-2'-0-Me-(CAGCAUCUAGCUACG)-3'] (SEQ ID NO: 3), RNA31 (s'-AUGUGGAUUGGCGAUAAAAAACAACU-GCUGU-3', fragment of lacZ mRNA from 2302 to 2331 with an 3'-overhang U) (SEQ ID NO: 4), DNA-2'-0-Me- RNA30.5 [5'-d(CAGCAGTTGTTTTT-T)-2'-Me-ribo(AUCG-CCAAUCCACAU)-3', complementary to lacZ mRNA from 2305-2334 nt,] (SEQ ID NO: 5), and DNA-2'-0-0-Me
  • GGAGAGUAUGCAGUAGUCAUCGCGACGUAGCUAGAUG-CUGAUUCAACUAC-3' was prepared by in vitro transcription of synthetic oligodeoxynucleotide templates with T7 RNA polymerase.
  • the above DNAs and RNA were purified by gel electrophoresis.
  • IPTG Isopropyl-.beta.-D-l-thiogalactopyranoside
  • DNA polymerase I was purchased from New England Biolabs; RNase H was purchased from GIBCO BRL. [a-32P]-dATP was purchased from NEN (PerkinElmer).
  • RNase H digestion reactions (5 ⁇ ) were generally carried out at 37. degree. C. for 1 hr in buffer [20 mM Tris-HCl (pH 7.5), 100 mM KC1, 10 mM MgCl.sub.2, 0.1 mM DTT, and 5% (w/v) sucrose], with RNA (1 pM-10 nM), DNA template or DNA-2'-0-Me- RNA hybrid template (1-500 nM), and RNase H (0.4 U/ ⁇ ). After ethanol precipitation of the digested RNA-DNA hybrid, Klenow extension was conducted. Klenow extension reactions (5 ⁇ ) were generally performed at 37. degree. C.
  • eukaryotic mRNA transcripts generally comprise a poly(A) tail and 3'- untranslated region (3'-UTR)
  • 3'-UTR 3'- untranslated region
  • RNase H digestion protocol was developed with which to remove the 3 -'region of a specific RNA transcript. Since RNase H is capable of digesting RNA which has formed a duplex with a DNA sequence (Nakamura and Oda. (1991) Proc. Natl. Acad. Sci. USA 88:11535-11539), the poly(A) tail and 3'-UTR can be removed by RNase H digestion after formation of such an RNA/DNA duplex. As previously reported, a 2'-methylated RNA sequence can bind to RNA and form a stable RNA/RNA duplex and the formation of the duplex provides a mechanism for protecting the bound RNA from RNase H digestion (Hayase et al.
  • RNase H recognizes the RNA/DNA duplex region and digests the RNA strand of the duplex.
  • Klenow recognizes the 2'-0- Me-RNA-DNA hybrid as a template and is capable of catalyzing a nucleotide extension reaction on the hybrid template.
  • the extension process on a hybrid template is shown to be as efficient as that observed on a non-hybrid, "regular" DNA template.
  • cleavage of the common 3 '-region of eukaryotic mRNAs is required to expose intrinsic internal sequences for selective RNA labeling and detection.
  • the 3 '-region of a test mRNA (lacZ mRNA) has been selectively removed to expose its internal sequences.
  • the sequence of the hybrid template for lacZ mRNA labeling and detection was designed based on the coding region of the target RNA, which is publicly available via GenBank (http://www.ncbi.nlm.nih.gov/).
  • RNA sequence 25-50 nt.
  • the selected template for the lacZ mRNA labeling and detection is DNA-2'-0-Me- RNA30.5 hybrid template [5'-d(CAGCAGTTGTTTTTT)-2'-ME- ribo(AUCGCCAAUCCAC-AU)-3'] (SEQ ID NO: 5), which is complementary to lacZ mRNA from nucleotide positions 2305-2334. Of which, six bases from nucleotide 2320-2325 are all adenine OA's). These A s served as the template for multiple rounds of a-32P-dATP incorporation during subsequent Klenow extension steps, which followed RNase H digestion to remove the 3 '-region of lacZ mRNA.
  • a lacZ-expressing plasmid is introduced into yeast.
  • the expression of lacZ from this plasmid is controlled by a galactose (Gal) promoter which can be induced in response to the presence of galactose in the media.
  • the promoter is not induced in the presence of glucose, which serves as an experimental negative control condition.
  • Two total mRNA samples were prepared: one sample was derived from galactose-induced yeast comprising the lacZ-expressing plasmid and a second sample was derived from yeast comprising the lacZ-expressing plasmid which were maintained in glucose-containing media, in the absence of galactose.
  • LacZ mRNA is selectively labeled and detected in IPTG-induced E. coli total RNA, as evidenced by a labeled lacZ mRNA large fragment (>2300 nt.) of the expected size. See FIG. 2, Lane 2. As expected, in the absence of Klenow, no mRNA is labeled (FIG. 2, Lane 4).
  • RNA31 (5' - AUGUGGAUUGGCGAUAAAAAACAACUG-CUGU-3 ' , fragment of lacZ mRNA from 2302 to 2331 with a 3 '-overhang U) (SEQ ID NO: 4) on DNA-2' -O-Me- RNA35.1 [5 ' -d(GTTGTTTTTT)-2' -Me-ribo(AUCGCCAAUCCAC AU)-d(CTCTGAA- AGA)-3' (SEQ ID NO: 6), complementary to lacZ mRNA from 2292 to 2326 nt]. See FIG. 3.
  • This double-digestion approach is also capable of detecting mRNA fragments, as well as full- length mRNA. Therefore, mRNA fragments arising from degradation can also be assayed using the method of the present invention, which further increases the detection sensitivity.
  • the ability to detect even degraded RNA illustrates yet another significant advantage of the present method over previously described methods for indirectly detecting RNA. Since E. coli and yeast comprise thousands of mRNA species (Rhodius et al. (2002) Annu. Rev. Microbiol. 56:599-624; Ross-Macdonald et al. (1999) Nature 402:413-418), the experimental results presented herein also underscore the selectivity of the present invention even in the presence of a plurality of other mRNA transcripts.
  • the method of the present invention has been used to selectively label and detect a specific mRNA transcript (i.e., LacZ mRNA) in a total RNA sample comprising thousands of different RNA transcripts. Furthermore, since only a small quantity of total RNA (0.4 ⁇ g) is used in each experiment (FIG. 2, Lanes 2-4), the method exhibits a high degree of sensitivity with regard to labeling and detection. 2. Discussion
  • RNA quantification As shown herein for the first time a novel method that combines RNase H cleavage and Klenow labeling to selectively label and detect a specific RNA (e.g., mRNA) in a total RNA sample have been developed and used. This direct and rapid RNA detection method has great potential for RNA quantification, especially individual mRNA
  • the method of the present invention provides significant experimental advantages over microarray and real-time PCR technologies.
  • the method of the present invention is complementary to conventional RNA detection methods, such as Northern blotting. Indeed, because this method for specific mRNA labeling allows assay of both fragmented and full-length mRNA, it greatly advances studies of mRNA decay and metabolic regulation. Total RNA, rather than mRNA, can be used for such labeling and detection studies, thereby obviating the need for mRNA isolation procedures which can result in degradation.
  • the present method is also compatible with the use of non-radioactive labels, such as fluorophore and antigen labels (Freeman et al. 1999, supra).
  • Such labels can be incorporated, and the labeling and detection determined by standard approaches, including the use of conjugated alkaline phosphatase or peroxidase to catalyze chemiluminescence reactions and ELISA quantitation (Young et al. (2002) J. Virol. Methods 103:27-39).
  • Example VI describes an ELISA procedure for use with the present method and chip.
  • the DNA-2'-0-Me-RNA hybrid template system has been developed, which enables RNase H and Klenow to share the same template and buffer. This methodological feature shortens the number of experimental steps and reduces the time required to obtain results. Gel electrophoresis can also be avoided by immobilizing the template on solid supports, such as a microplate or microchip surface (Benters et al. (2002) Nucl. Acids. Res. 30:el0). After RNA substrate immobilization, RNase H, Klenow, non- incorporated labels, and buffers can simply be washed away after each step.
  • the multi-label incorporation system is extremely useful for enhancing the detection sensitivity of the method, especially on solid phase or for long RNA transcripts analyzed by gel electrophoresis. See Examples III, IV and VIII for additional details.
  • RNase H digestion can remove mRNA 3 '-common sequences, such as a eukaryotic mRNA 3 '-poly (A) tail and 3'-UTR, this method is especially useful for direct mRNA labeling and detection in total RNA without the intricacies of reverse transcription and PCR procedures. This feature of the present method significantly simplifies the experimental procedure and shortens analysis time.
  • the RNA labeling method is highly sensitive and allows detection of RNA at attomole levels.
  • the detection sensitivity can be further enhanced by extending the length of the over-hang sequence of the DNA template.
  • the use of ELISA and micro-spotting techniques in conjunction with the present method also serves to increase the detection sensitivity.
  • the present method is also extraordinarly selective, as demonstrated by selective labeling and detection of lacZ mRNA in the presence of thousands of mRNAs. Therefore, as described herein below, this method can be used to advantage in microplate-based rapid and high-throughput detection technology, and in microchip-based rapid gene expression profiling technology.
  • RNAs expressed uniquely in each organism and which can be used as positive indicators of a contaminant (e.g., a pathogen) in a sample is of paramount importance in such settings.
  • Contaminant specific RNAs or "fingerprint" RNAs may include, without limitation, mRNA, ribosomal RNA, heteronuclear RNA, and mitochondrial RNA. Indeed, the expression profile of fingerprint RNAs is a powerful tool useful in the
  • identification of fingerprint RNAs using this rapid, sensitive, and selective strategy can lead to identification of microorganisms (such as pathogens), diseases, and/or characterization of disease status. This feature of the invention is described in greater detail elsewhere in the specification.
  • the method of the present invention has been modified for use with a solid matrix.
  • a solid matrix In order to further increase detection sensitivity, simplify the detection procedure, and avoid using radioactive material, fluorophore and enzyme labels were evaluated after template immobilization on a solid phase.
  • Solid matrices envisioned for use in the present invention include, without limitation, 96-well plates and microchips. It should be understood, however, that a variety of solid matrices are known in the art and may be used in the method of the invention.
  • the protocol developed for using fluorophore labeling in the present invention is similar to that used for radioactive labeling, but fluorophore-labeled dNTPs are used instead of radioactively labeled a-32P-dNTPs.
  • fluorophore-labeled dNTPs are used instead of radioactively labeled a-32P-dNTPs.
  • Enzyme labeling offers much greater sensitivity than the fluorophore labeling. This finding was likely a result of signal amplification that occurs during the course of an enzyme catalyzed reaction, such as that mediated by alkaline phosphatase. See FIG. 4.
  • the chemiluminescence detection may be performed by microplate luminometer, imaging system, or film detection. Although it is possible to detect RNA with fluorophore labels, the greater sensitivity observed with enzymatic labeling presents this approach as the exemplary labeling method at the present time. Moreover, chemiluminescence detection is simpler and requires less sophisticated equipment, attributes which further underscore its utility. For all of these reasons, additional experiments were performed using enzymatic labeling methodology.
  • LacZ mRNA was used as a test model RNA with which to evaluate the method of the present invention on solid phase. See FIG. 2 for schematic. After incubation of total mRNA sample in a 96- well plate (DNA-Bind.TM., purchased from Corning) on which the lacZ-mRNA hybrid template [5 '-d(CAGCAGTTGTTTTTT)-2'-Me-ribo(AUCGCCAAU- CCACAU)-NH.sub.2-3', binding to the lacZ mRNA from 2305-2334 nt] (SEQ ID NO: 5) had been previously immobilized, lacZ mRNA was bound specifically to the plate via the hybrid template and unbound mRNAs were washed away.
  • DNA-Bind.TM. purchased from Corning
  • RNA24 [ 5' - AUGUGGAUUGGCGAUAAAAAACAA-3 ' (SEQ ID NO: 8), the lacZ mRNA sequence from 2305-2328 nt.] was chemically synthesized and served as a positive control for the experiment; the underlined sequence is the binding region, and the italicized sequence is the digestible RNA-DNA duplex region.
  • RNA24 As anticipated, the positive control RNA24 was detected on the solid phase, whereas the negative control (no RNA) produced a signal not distinguishable from background levels. Consistent with the specific detection achieved using the method of the invention in solution (see Example II), lacZ mRNA present in galactose-induced cultures was specifically detectable using the present method in the context of presentation on a solid matrix. The minimal levels of lacZ mRNA present in glucose cultures (due to leaky expression) were also detectable, but at a level not significantly above background levels. Addition of RNA24 (positive control RNA) to the glucose sample, however, produced a strong signal, indicating that the presence of non-specific RNA in a sample does not interfere with detection of a specific RNA.
  • microchips This new strategy dramatically advances the ability to detect infectious disease, diagnose disease, and analyze gene expression patterns.
  • the method of the present invention also provides a powerful new tool for the biodefense arsenal.
  • FIG. 6 The general experimental flowchart involved in utilizing the present invention in the context of a solid matrix is shown in FIG. 6. Not only is the microtiter plate platform inexpensive to develop and manufacture, but it also benefits from its ready applicability to high-throughput screening and profiling. The following guidelines are set forth to illustrate how the methods of the invention may be used to design and develop RNA microchip technology.
  • Templates for example hybrid templates as described herein above, are first immobilized on a microchip, after which an RNA sample is added to the microchip matrix and incubated. After washing to remove unbound RNA, bound RNA is digested by RNase H to expose internal intrinsic sequences and then extended by Klenow DNA polymerase to incorporate antigen-labeled dNTPs. Bound RNA is subsequently labeled with enzymes by treating the microchip with antibody-enzyme conjugate. After washing the microchip to remove the non-specifically bound enzymes, substrate is added to generate chemiluminescent signals.
  • RNA microarray technologies have demonstrated that the detection of emitted fluorescent light from microspots or even nanospots is possible using a microarray reader (scanner) or high-resolution imaging system (Lockhart and Winzeler, 2000, supra; Trottier et al., 2002, supra), the detection of chemiluminescent light emitted from microspots on a RNA microchip is well within the capabilities of imaging technology.
  • the distance between the template-containing microspots on the RNA microchip should be large enough to prevent spot-to-spot interference during RNA sample binding, enzymatic steps, and chemiluminescent signal detection.
  • the microchip may be designed and prepared using glass chips (2.2.times.2.2 cm) surface-functionalized with COOH functional groups, which can be activated with N- hydroxylsuccinimide (NHS) for coupling with templates (e.g., hybrid templates) comprising 3'-terminal N3 ⁇ 4 groups.
  • templates e.g., hybrid templates
  • Such preparations may be achieved using established protocols known in the art (Zhou and Huang. (1993) Indian Journal of Chemistry 32B:35-39; Zhao et al. (2001) Nucleic Acids Res. 29:955-959; Manning et al. (2003) Materials Science and Engineering C 23:347-351).
  • gold-coated glass chips can be utilized for microchip preparation (Medalia et al. (2002) Ultramicroscopy 90:103-112; Fan et al. (2001) J. Am. Chem. Soc. 123:2454-2455; Hopfner et al. (1999) Applied Surface Science 152:259- 265).
  • RNA microchip To demonstrate direct RNA detection on a microchip, a low-density chip containing 16 templates on an area of 1.6 x 1.6 centimeters may be prepared. Subsequently, a microchip comprising 144 different templates on an area of 1.2 x 1.2 centimeters may be prepared using a microarrayer. It is anticipated that spot size on such a microchip will be approximately 600 ⁇ in diameter and the spot-spot gap approximately 400 ⁇ . If, for example, eighteen fingerprint RNAs are assessed for each microorganism, it is possible to monitor eight microorganisms simultaneously on a single microchip. Eight non-pathogenic microorganisms, including bacteria, viruses, yeasts, and fungi, may also be evaluated using the RNA microchip technology.
  • Eight sets of eighteen fingerprint RNAs may be chosen based on their high expression levels, a determination of which can be obtained via gene expression profiling. Such genes are good positive indicators for the presence of the microorganism in question. Such gene expression profiles can be purchased (Invitrogen, for example provides such services), determined experimentally, or potentially identified by reviewing the scientific literature germane to the microorganisms to be detected. A skilled artisan would be aware of these approaches and such considerations would be well within his/her capabilities.
  • the immobilized templates e.g., hybrid templates
  • immobilized on the microchip are designed based on the fingerprint RNAs, and each spot on the microchip can represent a different template recognizing a different fingerprint RNA.
  • Gene expression profiles of cells or organisms, including pathogens, may vary due to cell cycle stage, nutrient availability, and/or environmental conditions.
  • 100 fingerprint RNAs or more may be chosen from each organism or cell subtype to prevent misleading results associated with potential gene expression variation.
  • the long-term goal of this application of the method of the invention is to spot 10,000 templates on a microchip (2.5 x 2.5 cm), which would enable the detection of approximately 100 of the most virulent viruses, bacteria, and other pathogens.
  • microchips are ideally suited for biodefense applications and/or detection of pathogen-caused disease.
  • the RNA microchip technology may also be used in non-pathogenic disease analysis, disease classification, and microbial contaminant detection in food and/or water supplies for example.
  • hybrid templates are designed according to the following strategy.
  • a hybrid template e.g., 5'-DNA-2'-0-Me-RNA-3'
  • the 5'-DNA sequence allows RNase H to cleave in the 3' region of target RNAs prior to the Klenow extension step. This is a particularly important step when analyzing eukaryotic mRNA which generally comprise a 3'- poly(A) tail and 3'-UTR.
  • This design was demonstrated to be effective for the specific detection of lacZ mRNA in a large population of different RNA transcripts.
  • the successful execution of lacZ mRNA detection also demonstrates that the designed lengths of the regions of the LacZ hybrid template were sufficient to prevent adverse interactions between the solid matrix and the enzymes.
  • the 5 '-region of the target RNA can also be removed by the RNaseH digestion. See FIG. 6.
  • the template [5'-DNA-(2'-I-Me-RNA)-DNA-3'] is designed to contain three regions: a 5'-DNA sequence, a middle 2'-0-methyl-RNA sequence, and a 3'-DNA sequence. The 5'- and 3'-
  • DNA sequences allow RNase H to cleave both 3' and 5' regions of target RNAs, such as, for example, mRNAs. After the RNase H digestion and washing, only the target RNA fragment complementary to the 2'-0-Me-RNA sequence remains on the template, thus creating an RNA microchip for labeling and detection.
  • the 2'-0-Me-RNA region of the template is 4-78 nucleotides long, which provides sufficient sequence specificity while allowing stable RNA- RNA duplex formation capable of surviving the treatments involved in the present method.
  • the DNA regions of the template are 1-30 nucleotides long, which allows RNase H recognition of the RNA-DNA duplexes.
  • the sequence of the template is designed based on the fingerprint RNA sequence, which can be determined based on nucleic acid sequence data banks (e.g., GenBank), sequence projects, or genomic research.
  • the RNA sequence region (-6-80 nucleotides) used for the template design is chosen after examination of the RNA secondary structure using computer folding programs, such as Mfold (Genetics Computer Group, Madison, Wis.).
  • Mfold Genetics Computer Group, Madison, Wis.
  • the 5' -DNA region of the template which serves as a template for Klenow extension, can be any sequence.
  • RNA binding and Klenow extension it may be advantageous to use a three-region template [5'-DNA-(2'-0-Me-RNA)-DNA-3'], which allows RNase H cleavage of both 5' and 3' regions of target RNAs, and leaves just the complementary RNA fragment for labeling and detection.
  • the 3 '-termini of the templates is immobilized on the solid phase.
  • This arrangement allows the DNA polymerase to extend target RNA 3 '-ends on the hybrid templates.
  • Glass and polystyrene microchip functionalized with COOH or NH 2 groups, or gold plating can be used as the solid support.
  • the hybrid template may be immobilized on the microchip using several conventional systems (see Table I), including, (I) well-established protocols for immobilizing the 3'-NH 2 - template on a COOH-functionalized surface (Manning et al., 2003, supra).
  • an amino group (NH 2 ) is introduced into the 3 '-terminal of the template during solid phase synthesis, and this NH 2 group is coupled with the activated -COOH group (such as— CO— NOS).
  • the activated -COOH group such as— CO— NOS.
  • An alternate procedure which can be used to immobilize the template on an NH 2 - functionalized surface involves the introduction of a ribonucleotide residue into the 3'- terminal of the template during solid phase synthesis. The diol functionality on this residue is converted to two aldehyde functional groups by NaIO.sub.4 oxidation, prior to coupling with the amino group on the solid surface (Lemaitre et al. (1987) Proc Natl Acad Sci USA 84:648- 652).
  • a sulfide- or selenide-modified template can be immobilized on a gold surface based on a number of strategies known in the art (Medalia et al., 2002, supra;
  • the surface of the microchip may be capped with a variety of capping reagents. See Table 1. Such protocols are known to skilled artisans familiar with experimental variations designed to investigate the positive, negative, or neutral surface best suited for minimizing background noise. For instance, after immobilization of the sulfide- or selenide-modified templates on a gold surface, sulfide- or selenide-containing reagents are generally used to saturate the surface, which prevents sulfide and mercapto functionalities of the enzyme from binding to the gold surface.
  • RNase H digestion is used to remove the 3 '-region of target RNA thereby exposing internal intrinsic sequences for Klenow extension. Unlike site specific cleavage of DNA sequences, which is routine, site specific cleavage of RNA sequences is technically challenging. It has, however, been reported that RNase H is able to digest RNA strands when bound to DNA sequences. Although RNase H cleaves RNA non- specifically with regard to sequence, a bound DNA sequence can serve as a guide that directs RNase H to digest a specific region of RNA (i.e., the DNA bound region). Thus, the bound DNA transforms RNase H into a site-specific RNA endonuclease.
  • the present inventor has developed an approach to remove the 3 '-region of target RNA, including the poly(A) tail and 3'-UTR located in the 3'-region of most eukaryotic mRNAs.
  • a DNA-RNA hybrid template is designed to facilitate use of the same template for both RNase H digestion and Klenow extension.
  • both RNaseH and Klenow DNA polymerase recognize the hybrid 5'-DNA-2'-0-Me-RNA-3' templates.
  • Klenow polymerase recognizes the hybrid template of the invention as well as a DNA template.
  • a hybrid template 5'-DNA-2'- O-Me-RNA-3' comprising 5' DNA and 3' 2'-0-methylated-RNA sequences, allows RNase H to cleave the 3' region of target RNAs prior to the Klenow extension step.
  • the hybrid template 5'-DNA-(2'-0-Me-RNA)-DNA-3' comprising 5'-DNA, middle 2'-0-methyl-RNA, and 3'-DNA sequences, enables RNase H to cleave both 3' and 5' regions of target RNAs.
  • target RNA fragment complementary to the 2'-0-Me-RNA sequence remains on the template.
  • Such bound target RNA fragments are, therefore, available for Klenow extension and are consequently "tagged" for detection by incorporation of labeled nucleotide.
  • template immobilization is enzymatically compatible with both RNase H and Klenow polymerase activity. As described herein, reaction conditions compatible with the RNase H digestion and Klenow extension were developed that enabled these reactions to be performed simultaneously.
  • the disclosed methods are compatible with a variety of labeling systems, including but not limited to radioactive labeling, fluorophore labeling, and enzyme labeling.
  • Enzyme labeling was chosen as a preferred labeling system because it is sensitive, safe and accessible method (Pollard-Knight et al. 1990, supra; Reddy et al., 1999, supra).
  • Klenow polymerase mediated extension may be used to integrate antigen labels via the incorporation of antigen-labeled-dNTPs, such as 12-biotin-dATP.
  • the length of the 5'- region DNA sequence of the template may be used to control the number of the antigen- dNTPs incorporated into the bound RNAs (Huang and Szostak. (1996) Nucleic Acids
  • the 5'-DNA region of the template can be any sequence.
  • the antigen labeling is converted to enzyme labeling via treating a chip, for example, with an antibody-enzyme conjugate, such as an anti-biotin antibody- alkaline phosphatase conjugate.
  • an antibody-enzyme conjugate such as an anti-biotin antibody- alkaline phosphatase conjugate.
  • various aspects of the reaction can be varied, including the length of the 5 '-region DNA of the template, antigen linker size, and the purity of the antibody-enzyme conjugate. Such considerations are well known in the art and familiar to skilled artisans.
  • Systems that utilize small molecules and binder-enzyme conjugates, such as biotin and avidin- alkaline phosphatase conjugates are also envisioned as compatible with the method of the present invention.
  • RNA microchip of the present invention can be placed in a chamber, which facilitates supplementation with fresh substrate.
  • RNA microarray reader scanner
  • chemiluminescent detection sensitivity of this RNA direct detection system is comparable with the real-time PCR.
  • RNA detection signal produced by the present method is amplified through the enzyme-catalyzed reaction (Pollard-Knight et al. 1990, supra; Reddy et al., 1999, supra), enzymatic labeling of target RNAs offers high sensitivity.
  • the present inventor has determined that the detection sensitivity of alkaline phosphatase RNA labeling may reach as high as 10.sup.-22 moles on a microspot using the dioxetane substrate. Thus, approximately one hundred alkaline phosphatase molecules are detectable. If every bound RNA is labeled on average with several enzyme molecules, therefore, dozens of the target RNA are detectable.
  • RNA microchip detection does not suffer from similar partial degradation problems.
  • RNA chip of the invention Sixteen designed templates with 3'-NH2 groups may be immobilized on sixteen DNA-binding spots (each one 2.5 mm in diameter) on a glass microchip (1.6 x 1.6 cm) activated with NHS groups. To each DNA-binding spot, 1 ⁇ L ⁇ of coupling buffer (2x) and 1 ⁇ L ⁇ of the template (1 pmole) are added. After the chip is incubated for 0.5 hour at 37° C, the chip is washed with post-coupling washing buffer (3 x 1 mL) to remove the unbound templates. After heat denaturing, 1 of RNA sample is added to the SSC buffer (200 ⁇ ).
  • RNA is removed by washing the chip three times, each time with 1 mL of SSC buffer.
  • RNase H 2 U/ ⁇
  • RNase H buffer 200 uL
  • a solution of 1 ⁇ of Klenow (5 U/pL), 1 ⁇ . of dATP-Biotin (50 mM), and Klenow buffer 200 uL is added to the chip surface, and the chip is incubated with shaking for 15 minutes at 37° C.
  • the substrate may be re-added or supplemented continuously to maintain a steady signal emission.
  • the compatibility of an RNA microchip of the invention with manipulations in a chamber facilitates such substrate supplementation.
  • the protocol for the microchip containing 100 templates is analogous to the protocol described here.
  • the blocking buffer, washing buffer, and alkaline phosphatase buffer (lO.times.) are available commercially (Sigma). Other solutions are as follows: Coupling Buffer (lO.times.): 50 mM Na 2 HP0 4 .
  • Coupling buffer (10 ⁇ , 50 mM Na 2 HP0 4 , 10 mM EDTA, pH 9.0), RNase-free water (89 ⁇ ), and the 3'-NH.sub.2-template (1 ⁇ , 0.1-0.6 mM) is added to the DNA- binding 96-well plate (Corning), and the plate is incubated for one hour at 37° C. Each well is then washed three times with post-coupling washing buffer (250 ⁇ , 150 mM NaCl, 100 mM Maleate, pH 7.5) to remove the non-immobilized templates. ii. RNA binding and washing
  • RNase H buffer [50 ⁇ , 50 mM Tris-HCl (ph 7.5), 40 mM KC1, 6 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA]
  • RNase H (1.0 ⁇ , 0.2 units/uL) is added to each well, followed by 30 minute incubation at 37° C.
  • Klenow buffer [50 ⁇ , 10 mM Tris-Cl (pH 7.5), 5 mM MgCl 2 , 7.5 mM DTT] is added to each well, followed by addition of Klenow fragment (1 ⁇ , 5 units ⁇ L) and Biotin-7-dATP (1 ⁇ , 1 mM).
  • the plate is incubated for 1 hour at 37° C. Subsequently, the unincorporated biotin-dATP is removed from each well by washing twice with blocking buffer (250 ⁇ , lx, Sigma). Moreover, blocking buffer (250 ⁇ , 5x, Sigma) is used to wash each well.
  • blocking buffer 100 ⁇ L ⁇ , lx, Sigma
  • antibiotin-AP conjugate [1 ⁇ L ⁇ , 300 fold-diluted conjugate with blocking buffer (lx, Sigma)].
  • the plate is then incubated for 20 minutes at room temperature. After the incubation, each well is washed 4 times with washing buffer (250 ⁇ , lx, Sigma) and once with alkaline phosphatase buffer (250 ⁇ , lx, Sigma).
  • the CDP substrate 90 ⁇ , Sigma
  • alkaline phosphatase buffer 10 ⁇ L ⁇ , lOx, Sigma
  • mRNA with a 3'-poly(A) can be labeled and detected in a total RNA sample using a poly(T) template [Huang and Szostak (2003) supra] labeling and detection of a specific mRNA transcript has heretofore proven challenging due to shared 3 '-sequences, such as the 3 '-untranslated region (3'-UTR) and 3'-poly(A) tail of mRNA transcripts of eukaryotic organisms.
  • its 3 '-region is preferably removed to expose its unique internal sequences for selective labeling and detection.
  • RNA endonucleases capable of selectively cutting RNA are not readily available.
  • the present inventor has, however, discovered that RNase H can be used as an "RNA endonuclease" in the presence of a DNA guiding sequence since RNase H is capable of cutting RNA in an RNA/DNA duplex [Nakamura and Oda (1991) supra; Hayase et al. (1990) supra].
  • RNA/2 ' -Me-RNA duplexes An additional level of control is accorded by the enzymatic properties of RNase H, which is not capable of digesting RNA/RNA duplexes, including RNA/2 ' -Me-RNA duplexes [Nakamura and Oda (1991) supra; Hayase et al. (1990) supra].
  • the present inventor designed a 5'-DNA-(2'-Me-RNA)-3' hybrid template, wherein the DNA and RNA sequences serve as a guiding sequence and a protecting sequence, respectively.
  • the 5'-DNA sequence also serves as the template for Klenow extension.
  • the size of the template should be sufficiently long. Experiments by the present inventor show that a 10 nucleotide 3' -DNA sequence facilitates effective removal of the RNA 5 '-region by RNase H.
  • the hybrid template can be immobilized through a 3'-N3 ⁇ 4 group on a microplate via N-hydroxylsuccinimide (NHS) displacement to produce a functionalized microplate
  • RNA-binder conjugate e.g., anti-biotin antibody-alkaline phosphate (AP) conjugate
  • AP anti-biotin antibody-alkaline phosphate
  • An immobilized enzyme may be capable of, for example, catalyzing a chemiluminescence reaction in the presence of substrates (e.g., a dioxetane substrate) [Young et al. (2002) supra], which allows detection of a specific bound RNA.
  • substrates e.g., a dioxetane substrate
  • the signal detected in the present system is amplified via enzyme- catalyzed substrate turnover [Saghatelian et al. J. Am. Chem. Soc. 2003, 125, 344-345; Liu et al. J. Am. Chem. Soc. 2003, 125, 6642-6643].
  • RNA24.1 (5 ' - AUGUGGAUUGGCGAUAAAAAAC AA-3 ' (SEQ ID NO: 8), a section of the lacZ mRNA sequence) is used as the target RNA, and DNA35.2 [5'-d(GTTGTTTTTT)-2'-Me-RNA(AUCGCCAAUCCACAU)-d(CTGTGAAAGA)-NH 2 -3'] (SEQ ID NO: 9) is utilized as the template for RNA 24.1 and the double digestion template for lacZ mRNA.
  • SEQ ID NO: 8 DNA35.2
  • RNA detection sensitivity can reach as high as 1 fmole (10-15 mole) of RNA (FIG. 15B).
  • FIG. 15B the film was exposed on the microplate for five hours after the dioxetane substrate addition.
  • background signals also increase.
  • signal due to background is reduced with shorter exposure times (FIG. 15A).
  • the signal/noise ratio and sensitivity can be significantly increased using smaller micro-well plates or microchips.
  • the washing steps were increased to reduce the amount of non-specifically bound conjugate.
  • washing steps can be altered to change the number of washing cycles and/or the stringency of the wash conditions.
  • Other approaches such as protein blocking and chemical coating [Stratis- Cullum et al. Anal. Chem. 2003, 75, 275-280], can also reduce and/or prevent non-specific sticking of enzyme conjugates.
  • Other adaptations useful for optimizing the present invention with regard to a preferred signal/noise ratio and desired sensitivity are also known to a skilled artisan.
  • yeast mRNA samples were prepared.
  • One sample contains lacZ mRNA isolated from a yeast strain (CWXY2) containing galactose-inducible lacZ-expressing plasmids (PEG202/Ras, PJG4-5/Raf, pCWX24) [Xu et al. Proc. Natl. Acad. Sci. USA 1997, 94, 12473-12478; Huang and Alsaidi, Analytical Biochemistry, 2003, 322, 269-274], and the other is isolated from glucose-repressed cells that do not express lacZ mRNA [Barkley and Bourgeois, 1978, supra; Khodursky et al.
  • the present inventor has developed a novel system for specific RNA detection on a microplate by immobilizing the hybrid templates and using enzyme labeling for detection (e.g., AP).
  • enzyme labeling for detection e.g., AP
  • the system of the present invention is direct, simple, cost-effective and rapid and does not require reverse transcription, PCR, transcription, laser excitation and fluorescence detection.
  • This method is extremelyly selective, in that only lacZ mRNA was specifically detected among all of the mRNA molecules present in the pool of cellular RNA transcripts, and sensitive, exhibiting an ability to detect a specific RNA at the fmole level.
  • the detection sensitivity can be further increased by using a smaller plate or a microchip (see Example IV for details). Moreover, experimental time and steps are further reduced when the present system involves utilization of a microchip as a solid phase. Reduction of background signals can also be used effectively to increase the detection sensitivity [Stratis-Cullum et al. 2003, supra]. [0170]
  • the present method is also particularly well suited to analyses of environmental samples, wherein mRNAs are frequently present in a partially degraded state, since only a short portion of an mRNA molecule is needed for detection in this method. This novel strategy has great potential for use in rapid on-site detection of bacteria and viruses via identification of their signature RNAs. As indicated herein above, this strategy is applicable to RNA microarray technology achieved by systematic template immobilization on microchips. This approach facilitates rapid detection of pathogens and diseases in emergency situations, for point-of-care diagnosis, and for direct gene expression profiling.
  • Example VI RNA Microchip for Rapid, Direct and Specific Detection of Biological RNA
  • RNA detection is essential for monitoring, prevention and control of pathogen-caused epidemics.
  • Disclosed herein is a novel RNA microchip strategy, which can rapidly and directly detect any RNA sequences without RT-PCR and transcription amplification. Based on the DNA lagging synthesis in the gene replication, this simple approach is developed via DNA polymerase polymerization of a RNA primer on a DNA template and RNase H digestion of RNA on a DNA guide to expose unique sequence for specific detection.
  • the chimeric probe [5'-DNA-(2'-Me-RNA)-DNA-3'] is specifically designed to allow RNase H digestion and DNA polymerase extension on the same detecting probe.
  • Biotin-labeled dNTPs are incorporated by DNA polymerase to the targets for direct RNA detection with chemiluminescent signal by a sensitive CCD camera.
  • RNA microchip spot size: 75 micron
  • single-nucleotide specificity, high sensitivity (at the low fmole level), and rapidness (approximately 20-min detection time) have been demonstrated.
  • direct detection of a specific RNA from a biological sample has been achieved, without the need for the time-consuming amplification and hybridization steps.
  • the rapid and accurate RNA microchip technology can be effective in fast epidemic monitoring, field detection, clinical diagnosis, and food processing monitoring.
  • the present Example describes a rapid and accurate methodology for RNA direct detection in order to avoid the target amplification (such as RT-PCR) and fluorescent detection, which requires laser excitation.
  • RNA direct detection in order to avoid the target amplification (such as RT-PCR) and fluorescent detection, which requires laser excitation.
  • RNA microchip strategy for rapid and direct detection of specific RNAs, which is fundamentally different from the existing technologies. This simple approach is fast (approximately 20-min detection time), and has high specificity (single-nucleotide discrimination) and high sensitivity.
  • a specific mRNA has successfully been detected in E. coli total RNA.
  • the methodology takes advantage of the DNA lagging synthesis in the gene replication, [ Okazaki et al. 1969 ] where a DNA polymerase naturally extends a RNA primer with dNTPs on a DNA template.
  • DNA polymerase is used to incorporate labels directly into a target RNA on a DNA template immobilized on a microchip (Fig. 6).
  • the RNA Prior to the polymerase extension, the RNA is cleavage by RNase H, which opens an internal sequence of RNA (such as mRNA) for the specific extension of the hapten-labeled dNTPs (such as biotin- dNTPs)[Alsaidi et al. 2004; Spencer et al. 2010].
  • a binder- enzyme conjugate e.g., streptavidine-horse radish peroxidase
  • the hapten labels are subsequently converted to enzymatic labels that catalyze chemiluminescent reactions for detection by a CCD camera with high sensitivity [Ronaghi et al. 1998].
  • chimeric probes [5'-DNA-(2'-Me-RNA)- DNA-3', or 5'-DNA-(2'-Me-RNA)-3')] were designed and synthesized to consolidate the target RNA hybridization, RNase H digestion and DNA polymerase extension (Fig. 6 and
  • the 5' -DNA sequence serves as both the DNA guide for RNase H digestion and the DNA template for DNA polymerase extension.
  • the 3' -DNA sequence serves as another DNA guide for RNase H digestion to cleave 5 '-region of target RNAs. Since RNase H does not digest RNA/RNA duplex[Hayase et al. 1990], the 2'-Me-RNA portion in the chimeric probe prevents the excessive digestion of the target RNA, which retains the target on the probe for the DNA polymerization. The 2'-methylation of the probe RNA portion also stabilizes the probe RNA sequence against RNases.
  • RNAs with 3'-NH2 groups were immobilized on the NHS -activated silicon surface via microarrayer spotting with typical 75 micron in size.[Benters et al. 2002] Any RNAs can be specifically detected by designing complementary probes.
  • the novel RNA microchip technology has been successfully demonstrated herein by using biotin-labeled dATP and RNAs containing a few consecutive As (Fig. 22).
  • the hybridization step is the major rate-limiting step among DNA microchip technologies [Xiao et al. 2006, Rothlingshofer et al. 2008, Barken et al. 2007, Liu et al. 2003, Sakamoto et al. 2005], which usually requires overnight hybridization. Shorter oligonucleotides may hybridize to their complementary sequences faster than the longer ones. Since short target RNAs (normally 15-25 nt.) are used in the RNA microchip detection, fast kinetics of the target RNA/probe hybridization are expected.
  • RNA(AUCGCCAAUCCACAU)-d(CTGTGAAAGA-NH 2 -3'] was designed to target lacZ mRNA (the target fragment: 5'-AUGUGGAUUGGCGAUAAAAAACAA-3').
  • RNA microchip Using the RNA microchip, a high sensitivity for the RNA detection was demonstrated, and detected target RNA at the level as low as 1 fmol (Figure 19A).
  • lacZ mRNA was specifically detected in the total RNA isolated from wild- type E coli ( Figure 19B) grown in the presence of IPTG, which induces lacZ mRNA expression. Total RNA contains several thousands of different mRNAs. The chemiluminescent signal was not detected in the total RNA isolated from wild-type E coli.
  • RNA microchip strategy which can rapidly and directly detect specific RNAs without RT-PCR and transcription
  • RNA microchip spot size: 75 micron
  • the high sensitivity the low fmole level
  • This RNA microchip methodology is simple, and the entire detection can be completed in approximately 20 minutes.
  • RNA microchip technology well suited for point-of-care detection with a wide range of potential applications, such as fast epidemic monitoring, field detection, clinical diagnosis, and food processing monitoring.
  • RNA detection is essential for monitoring, prevention and control of pathogen-caused epidemics.
  • Figure 20 demonstrates a novel RNA detection strategy with the enzymatic reactions on RNA microchip (spot size: 75 micron), which offers single-nucleotide specificity, rapidness (approximately 20-min detection time), and high sensitivity (at the low fmole level), and which allows direct detection of specific RNAs from biological samples.
  • the silicon chips (0.5 x 0.5 cm) were first degreased by treatment in CH 2 CI 2 for 30 min with gentle shaking, followed by cleaning in concentrated H 2 SO 4 for 1 h. The chips were rinsed in RNase free water several times until the wash was at pH 7.0. The chips were covered with a mixture of 3% 3-aminopropyltrimethoxysilane (Aldrich, St. Louis, MO) in a ethanokwater (19:1) solution for 30 min at RT. The chips were washed sequentially with methanol and RNase free water and were dried at room temperature before activation.
  • Activation was performed by incubating in a solution containing 10 mM 1 ,4-phenylene diisothiocyanate (PDITC, Fluka, Buchs, Switzerland) in CH 2 CI 2 with 1% pyridine for 2 h.
  • PDITC 10 mM 1 ,4-phenylene diisothiocyanate
  • the synthesized DNA-RNA-DNA chimeric probe was diluted to 200 ⁇ in 100 mM sodium phosphate (pH 8.5) and was printed on the activated microchip (0.5 x 0.5 cm) with the OmniGrid Micro, followed by 30 min incubation in a water bath at 37 °C.
  • the microchips were incubated with 5X SSC buffer (3.0 M NaCl, 3.0 M sodium citrate, pH 7.0) and RNA samples at room temperature for 15 min. Subsequently, the unbound RNAs were removed by washing each chip three times with 2X SSC buffer (1.2 M NaCl, 1.2 M sodium citrate, pH 7.0).
  • the microchips were incubated with RNase H buffer (20 ⁇ L ⁇ , 1 X, New
  • the prepared microchips immobilized with the chimeric probes were incubated with 5X SSC buffer (3.0 M NaCl, 3.0 M sodium citrate, pH 7.0) and RNA samples at room temperature for 5 min. Subsequently, the unbound RNAs were removed by washing each chip three times with 2X SSC buffer (1.2 M NaCl, 1.2 M sodium citrate, pH 7.0). The chips were incubated with RNase H buffer (20 ⁇ , 1 X, New England BioLabs, Ipswich, MA) and RNase H (0.5 ⁇ , 5,000 u mL 1 , New England Biolabs), followed by 5 min incubation at 37 °C.
  • the microchip surface was rinsed with StartingBlock (TBS) Blocking Buffer (Pierce, Rockford, IL).
  • TBS StartingBlock
  • the chips were then incubated with Klenow buffer (20 ⁇ L ⁇ , 1 X, New England Biolabs), the Klenow fragment (0.5 ⁇ , 5,000 u mL "1 , New England Biolabs), and biotin-7-dATP (1.0 ⁇ , 0.4 mM Invitrogen, Carlsbad, CA) for 5 min at 37 °C. Subsequently, the unincorporated biotin-dATP was removed from each well by washing three times with StartingBlock (TBS) Blocking Buffer (Pierce).
  • the chips were incubated with Poly-HRP-streptavidin conjugate (Thermo Scientific, Rockford, IL) diluted 5,000x in StartingBlock (TBS) Blocking Buffer for 5 min. The chips were rinsed several times to remove unbound enzymes. SuperSignal ELISA Femto Maximum Sensitivity Substrate (Invitrogen) was added and the chemiluminescent signal was detected with a sensitive CCD camera.
  • the chimera probes were synthesized in our laboratory by using the standard DNA phosphoramidites and regular 2'-MeO-RNA phosphoramidites, which are
  • Swine Flu RNA [SF, Swine Influenza (H1N1) matrix protein 1 (Ml) mRNA: 44- 82 nt]: 5 '- AGAUCGCGCAGAGACUGGAAAGUGU-3 ' (SEQ ID NO:31)
  • lacZ mRNA (lacZ, E. coli lacZ mRNA: 724-748 nt): 5'- AUGUGGAUUGGCGAUAAAAAACAA-3 ' (SEQ ID NO:8)
  • lacZ probe 5'-d(GTTGTTTTTT)-2'-0-Me-RNA(AUCGCCAAUCCACAU)- d(CTGTGAAAGA)-NH 2 -3 ' (SEQ ID NO: 9)
  • Bacillus anthraces RNA (BA, B. anthraces lethal factor mRNA: 855-892 nt) : 5 ' - AUCUUUAGAAGCAUUAUCUGAAGAUAAGAAAAAAA-3 ' (SEQ ID NO:33)
  • BA Probe 5'-d(GATTTTTTT)-2'-0-Me-RNA(CUUAUCUUCAGAUAA)- d(TGCTTCTAAAGAT)-NH2-3 ' (SEQ ID NO:34)
  • BF Probe 5 ' -d(TCGTTTTT)-2 ' -Q-Me(GGUAGGUCUGC AAAAUUUU)- d(CAAGAAGATT)-NH 2 -3 ' (SEQ ID NO:36) [0192] Target RNA for hybridization kinetics : 5 ' -
  • RNA for specificity study [the Bacillus anthraces RNA (BA) is B. anthraces lethal factor mRNA: 855-892 nt] :
  • Probe 1 (BA1): 3'-d(TAGAAATCTTCGT)-2'-0-Me-RNA(AAUAGACUUCUAUUC)- d(TTTTTTT)-5' (SEQ ID NO:38)
  • Probe 2 (BA2): 3'-d(TAGAAATCTTCGT)-2'-0-Me-RNA(AAUAGCCUUCUAGUC)- d(TTTTTTT)-5' (SEQ ID NO:39)
  • Probe 3 (BA3): 3'-d(TAGAAATCTTCGT)-2'-0-Me-RNA(AAUAGACUUCUAUUC)- d(TTTTTTTAG)-5 ' (SEQ ID NO:40)
  • Probe 4 (BA4): 3'-d(TAGAAATCTTCGT)-2'-0-Me-RNA(AAUAGACUUCUAUUC)- d(TTTT ATTAG) - 5 ' (SEQ ID NO:41)
  • Probe 5 (BA5): 3'-d(TAGAAATCTTCGT)-2'-0-Me-RNA(AAUAGACUUCUAUUC)- d(TATTATTAG)-5' (SEQ ID NO:42) v.
  • Microscope, camera and imaging software
  • RNA detection on microchip was assembled with a microscope (Nikon Eclipse 80i) and an ultra-sensitive CCD camera (VersArray System from Princeton Instruments, Princeton, NJ) with a software (ImagePro Plus).
  • the RNA rapid detection is normally performed by using 2x2 image binning, 10-30 second exposure (usually 15 seconds), 2X lens, and object distance of 1.5 cm.
  • the synthesized DNA-RNA-DNA chimeric probe was diluted to 200 ⁇ in 100 mM sodium phosphate (pH 8.5) and was printed on the activated chip with the OmniGrid
  • RNA 500 fmol was allowed to hybridize with the chip-immobilized probes in 5X SSC buffer (20 ⁇ , 3.0 M NaCl, 3.0 M sodium citrate, pH 7.0) for the indicated amount of time (from 0 to 40 minutes). After the chip washing, the microchip was then incubated with Poly-HRP- streptavidin conjugate (Thermo Scientific, Rockford, IL) diluted 5,000x in StartingBlock
  • TBS Blocking Buffer for 15 min.
  • the microchip was rinsed three times to remove unbound enzymes.
  • SuperSignal ELISA Femto Maximum Sensitivity Substrate (Invitrogen) was finally added and the chemiluminescent signal was immediately detected with an ultra- sensitive CCD camera.
  • the synthesized DNA-RNA-DNA chimeric probe was diluted to 200 ⁇ in 100 mM sodium phosphate (pH 8.5) and was printed on the activated chip with the OmniGrid Micro, followed by 30 min incubation in a water bath at 37 °C. The chips were incubated with 5X SSC buffer (3.0 M NaCl, 3.0 M sodium citrate, pH 7.0) and RNA samples at room temperature for 15 min. Subsequently, the unbound RNAs were removed by washing each chip three times with 2X SSC buffer (1.2 M NaCl, 1.2 M sodium citrate, pH 7.0).
  • the chips were incubated with RNase H buffer (20 ⁇ ,, 1 X, New England BioLabs, Ipswich, MA) and RNase H (0.5 ⁇ ,, 5,000 u mL 1 , New England Biolabs), followed by 30 min incubation at temperatures (37-65 °C). After draining the RNase H solution from each incubation chamber, the surface was blocked with StartingBlock (TBS) Blocking Buffer (Pierce, Rockford, IL) for 20 min.
  • TBS StartingBlock
  • the chips were then incubated with Klenow buffer (20 ⁇ ,, 1 X, New England Biolabs), the Klenow fragment (0.5 ⁇ L ⁇ , 5,000 u mL "1 , New England Biolabs), and biotin-7- dATP (1.0 ⁇ , 0.4 mM Invitrogen, Carlsbad, CA) for 1 h at temperatures (37-65 °C).
  • Klenow buffer (20 ⁇ ,, 1 X, New England Biolabs
  • the Klenow fragment 0.5 ⁇ L ⁇ , 5,000 u mL "1 , New England Biolabs
  • biotin-7- dATP 1.0 ⁇ , 0.4 mM Invitrogen, Carlsbad, CA
  • the synthesized DNA-RNA-DNA chimeric probe was diluted to 200 ⁇ in 100 mM sodium phosphate (pH 8.5) and was printed on the activated chip with the Omni Grid Micro, followed by 30 min incubation in a water bath at 37 °C. The chips were incubated with 5X SSC buffer (3.0 M NaCl, 3.0 M sodium citrate, pH 7.0) and RNA samples at room temperature for 15 min. Subsequently, the unbound RNAs were removed by washing each chip three times with 2X SSC buffer (1.2 M NaCl, 1.2 M sodium citrate, pH 7.0).
  • the chips were incubated with RNase H buffer (20 ⁇ , 1 X, New England BioLabs, Ipswich, MA) and RNase H (0.5 ⁇ , 5,000 u mL "1 , New England Biolabs), followed by 30 min incubation at 37 °C. After draining the RNase H solution from each incubation chamber, the surface was blocked with StartingBlock (TBS) Blocking Buffer (Pierce, Rockford, IL) for 20 min. The chips were then incubated with Klenow buffer (20 ⁇ L ⁇ , 1 X, New England Biolabs), the
  • the hybridization buffer can comprise a salt, such as, for example, NaCl and sodium citrate.
  • the NaCl or sodium citrate can be at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 M, or any amount in between.
  • the pH of the hybridization buffer can be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, or any amount above, below, or in between.
  • a specific example includes the hybridization buffer at 3.0 M NaCl, 3.0 M sodium citrate, pH 7.0.
  • the RNase digestion buffer can be comprised of, for example, comprised of Tris- HC1, KC1, MgCl 2 , and/or dithiothreitol.
  • the Tris-HCl can be at 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM, or any amount above, below, or in between.
  • KC1 can be at 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM, or any amount above, below, or in between.
  • the MgCl 2 concentration can be 1, 2, 2.5, 3, 3.5, 4, or any amount above, below, or in between.
  • the dithiothreitol concentration can be at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM, or any amount above, below, or in-between.
  • the pH of the solution can be be be be
  • RNase H digestion buffer is 50 mM Tris-HCl, 75 mM KC1, 3 mM MgCl 2 , 10 mM Dithiothreitol, pH 8.3.
  • the Klenow buffer can be comprised of, for example, Tris-HCl, NaCl, MgCl 2 , and dithiothreitol.
  • the Tris-HCl can be at 0, 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM, or any amount above, below, or in between.
  • NaCl can be at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM, or any amount above, below, or in between.
  • the MgCl 2 concentration can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any amount above, below, or in between.
  • the dithiothreitol concentration can be at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM, or any amount above, below, or in- between.
  • the pH of the solution can be be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,
  • Klenow digestion buffer is 50 mM Tris-HCl, 75 mM KC1, 3 mM MgCl 2 , 10 mM
  • the RNase H and Klenow buffers can be mixed at a ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts Klenow buffer to 1, 2, 3, 4 ,5, 6, 7, 8, 9, or 10 parts RNase buffer, or any ratio above, below, or in between this range. In one specific example, they were mixed in a ratio of 8 parts Klenow buffer and 2 parts Rnase H buffer.
  • the temperature for these enzymatic reactions can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 28, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C or any amount above, below, or in between. In one example, the temperature was 37°C.
  • a cocktail was prepared containing 2.4 ⁇ . BA RNA (5 ⁇ ) 4 ⁇ . RNase H, 4 ⁇ . Klenonw, 8 ⁇ ⁇ -dATP 32 , and 2:8 RNase H : Klenow buffer (New England Biolabs).
  • 4 ⁇ of the cocktail was added to 1 ⁇ of hybrid probe (1 ⁇ ) and allowed to react for 60 min. After the polymerization reactions were completed, samples were cooled to room temperature. The samples were then diluted 1:1 with Gel loading dye (7.5 ⁇ , containing 0.1% bromphenol blue, 0.1% xylene cyanol, 100 mM EDTA, and saturated urea) and run on 15% PAGE and visualized by autoradiography
  • a cocktail was prepared containing ⁇ BA RNA (1 ⁇ ) 0.5 ⁇ RNase H, 0.5 ⁇ Klenonw, 0.5 ⁇ biotin-dATP (0.4 mM), and 20 ⁇ of 2:8 RNase H : Klenow buffer (New England Biolabs).
  • the cocktail was pipetted on top of a microchip with immobilized BA probes on the surface and allowed to react for 60 min at 37°C.
  • the microchip was then washed three times with StartingBlock(TBS) Blocking Buffer (Pierce).
  • the chips were incubated with Poly-HRP Streptavidin (Thermo Scientific, Rockford, IL) diluted 5,000x in StartingBlock (TBS) Blocking Buffer for 15 min.
  • the chips were rinsed several timed to remove unbound enzymes.
  • SuperSignal ELISA Femto Maximum Sensitivity Substrate (Invitrogen) was added and the chemiluminescent signal was detected with a CCD camera.
  • the present method can combine the hybridization, digestion and extension steps in a single buffer
  • the present method can, alternatively, combine these steps in a single reaction vessel wherein the hybridization buffer, the digestion buffer and the extension buffer described herein (e.g., Example VII) are added sequentially to the vessel to perform each step.
  • the reaction buffers can be provided sequentially to the reaction vessel with or without rinses between the addition of each buffer.
  • the RNA detection microchip can be used with very small quantities of buffer solution.
  • the RNA detection methods and chips described herein are usable in microfluidic devices.
  • the devices that are compatible with the present methods and chips are the following: (1) microarrays, as exhibited in U.S. Pat. No. 6,607,886 and U.S. Pat. No. 5,807,522, where each array has an RNA probe that is attached to a solid surface and specifically responsive to target analyte;
  • unit-volume microdroplets containing, for example, each of several buffers in our invention can move in confined microfluidic channels to unit cells of different RNA probes;

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Abstract

La présente invention concerne un procédé très sensible et spécifique de détection directe d'au moins un ARN spécifique dans un échantillon. La présence d'un ARN spécifique produit un indicateur positif d'un agent pathogène, d'un contaminant, et/ou de gènes ou produits géniques normaux ou anormaux dans l'échantillon. Des applications pour lesquelles le procédé de l'invention est particulièrement bien adapté comprennent le diagnostic pathologique dans des lieux fournissant des soins médicaux, la détection précoce du cancer, la détection d'une contamination microbienne dans des aliments et/ou des sources d'eau, et la détection d'agents pathogènes dans le domaine de la biodéfense.
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WO2015081088A1 (fr) * 2013-11-27 2015-06-04 Immucor, Gtt Diagnostics, Inc. Détection directe d'arn par polymérisation enzymatique initiée en surface
WO2018222907A1 (fr) * 2017-06-02 2018-12-06 The United States Of America As Represented By The Secretary Of The Navy Procédés de détection moléculaire à médiation par phage et aspects associés
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WO2014165814A1 (fr) * 2013-04-04 2014-10-09 Georgia State University Research Foundation, Inc. Détection par micropuce à arn utilisant l'amplification de signal assistée par nanoparticule
CN105283561A (zh) * 2013-04-04 2016-01-27 佐治亚州立大学研究基金会公司 使用纳米粒子辅助信号放大的rna微芯片检测
JP2016515827A (ja) * 2013-04-04 2016-06-02 ジョージア ステイト ユニバーシティ リサーチ ファンデーション， インコーポレイテッド ナノ粒子支援シグナル増幅を使用するｒｎａマイクロチップ検出
WO2015081088A1 (fr) * 2013-11-27 2015-06-04 Immucor, Gtt Diagnostics, Inc. Détection directe d'arn par polymérisation enzymatique initiée en surface
US11130987B2 (en) 2013-11-27 2021-09-28 Sentilus Holdco, Llc Direct detection of RNA by surface initiated enzymatic polymerization
WO2018222907A1 (fr) * 2017-06-02 2018-12-06 The United States Of America As Represented By The Secretary Of The Navy Procédés de détection moléculaire à médiation par phage et aspects associés
US20200263233A1 (en) * 2017-06-23 2020-08-20 Eiken Kagaku Kabushiki Kaisha Method For Nucleic Acid Detection, Primer For Nucleic Acid Detection, And Kit For Nucleic Acid Detection

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