KR100575407B1 - Enzyme-specific cleavable polynucleotide substrate and assay method - Google Patents

Abstract

The present invention provides a reagent comprising an enzyme specific cleavable polynucleotide (30, 31) substrate having a quenched fluorescent portion (32, 33), and also provides a method for preparing the same. The polynucleotides 30 and 31 comprise at least one fluorescent part 32 which is sufficiently close to the other fluorescent part 33 to substantially quench the fluorescence of the fluorescent parts 32 and 33, and the fluorescent part 32 , 33) can be easily detected by fluorescence measurement techniques when separated by cleavage of polynucleotides. In addition, there is also provided a biological analysis method comprising the step of combining the reagent with a test sample that may contain the enzyme (36) to be analyzed, wherein the enzyme (36) cleaves the polynucleotide to the fluorescent portion (32, 33) and yield an increase in fluorescence intensity. This assay can be used for microbial detection and identification, bactericidal confirmation, drug discovery, enzyme analysis, immunoassay and other biological assays.

Description

Enzyme-specific cleavable polynucleotide substrates and methods of analysis thereof {ENZYME-SPECIFIC CLEAVABLE POLYNUCLEOTIDE SUBSTRATE AND ASSAY METHOD}

The present invention relates to a reagent comprising an enzyme specific cleavable polynucleotide substrate having a quenched fluorescent moiety, a method for preparing the same and an analytical method using the same.

Nucleases (DNases and RNases) are enzymes that hydrolyze and cleave polynucleotides, ie nucleic acids such as DNA and RNA. Such enzymes are present in all biological cells and perform many important functions in nucleic acid metabolism, replication, recombination, repair and modification. Nucleases can be classified by their substrate specificity, some nucleases cleave both DNA and RNA, some other nucleases act only on one type of nucleic acid, and some others nucleases Specificity, others are not sequence specific, some nucleases act on single-stranded nucleic acids, and other nucleases act only on double-stranded DNA. In addition, nucleases may in particular be located at the position of the bond they cleave; Location on the cell, ie intracellular or extracellular location; Ionic and pH conditions; And depending on resistance to heat treatment. Nucleases are described in detail in Linn, SM, Lloyd, RS, Roberts, RJ, Nucleases (2 ^nd ed.), Cold Spring Harbor Laboratory Press, Plainview, NY, 1994.

Antibiotic resistant bacterial strains and foodborne diseases are a serious problem for the public. In addition, the management of the risk of microbial contamination in the healthcare, cosmetics, food and beverage industries is an important issue in terms of health and stability. Bacterial testing is an essential part of measuring microbial risk. Enzymes produced by bacteria are commonly used to test for the presence of bacteria. The action of microorganisms to hydrolyze and cleave nucleic acids through the production of nucleases has long been known. Enzymes are particularly useful for the detection of non-proliferative bacterial cells because they facilitate the cleavage reaction that cleaves up to millions of substrate molecules per second to detect a small number of bacterial cells. Certain nucleases produced by bacteria constitute a widely used means of identifying and identifying specific pathogenic species.

Known methods of measuring nuclease activity include UV absorption and electrophoresis or chromatographic separation of cleaved DNA fragments followed by fluorescence staining. As another method, the following methods using natural DNA, synthetic substrates and DNA binding molecules have been developed.

(i) colorimetric DNA binding molecules

Toluidine blue (TB) or methyl green (MG) can be added to the mixture of natural DNA and appropriate growth medium. This dye forms a complex with DNA that shows blue (TB) or green (MG). When the complex is hydrolyzed by DNase, it releases the dye, resulting in a color difference zone: rosy pink (TB) or colorless (MG).

(ii) fluorescent DNA binding molecules

Acridine orange (AO) or other suitable dyes such as hex dye 33258 or dimer cyanine nucleic acid dyes (e.g., Molecular Probes, Eugene, Oregon, Inc.) in a mixture of natural DNA and growth medium Products under the trade names TOTO ™ or YOYO ™) may be added. The compound shows weak fluorescence in aqueous solution but turns to strong fluorescence when bound to DNA. When DNA is hydrolyzed by DNase, the fluorescence is completely removed and can be identified as a non-fluorescent ring around all bacterial colonies.

(iii) chromogenic synthetic substrates

When detecting nuclease activity by this method, two kinds of synthetic substrates are used. One of them is thymidine 5'-monophosphate p-nitrophenyl ester ammonium salt. This releases p-nitrophenol upon hydrolysis by purified extracellular nucleases derived from Bacillus spp., Which is confirmed by an increase in absorbance at 410 nm. The other one is 5-bromo-4-chloro-indolylthymidine-3'-phosphate, which releases a doxylyl group upon hydrolysis to yield a bluish green indigo color by automatic oxidation.

(iv) fluorogenic substrates

There are two ways to do this. First is fluorescence measurement using poly (deoxy (etheno) adenylate) which is a derivative of poly (deoxyadenylate). This polynucleotide is inherently weak fluorescent. However, an increase in fluorescence is observed due to the release of fluorescent etenomononucleotides upon DNase hydrolysis (excitation = 320 nm and emission = 410 nm). This signal can be correlated with the amount of mononucleotides that are continuously monitored and released. As another method of fluorescence measurement, it is known to use a fluorescence "dequenching" technique. 14-mer polynucleotides may be labeled with Fluorescein 5-isothiocyanate (Ex = 490 nm and Em = 520 nm). When hybridized with unlabeled complementary strands, 50% of the fluorescence is quenched due to the interaction of the fluorophores with DNA. Cleavage, or chain cleavage, of the hybridized species by restriction endonucleases results in a twofold increase in fluorescence intensity due to “dequenching”.

DNA binding molecules are generally not sequence specific and thus the signal is affected by any endogenous DNA present in the sample. In some cases, it is also affected by the presence of surfactants, EDTA or cell debris. Chromatic synthetic substrates generally increase the cost of analysis because they require large amounts (millimolar), and in some cases may not dissolve. In addition, the adsorption-based method is several times less sensitive than the method based on fluorescence. With regard to fluorogenic substrates , few reagents are available for nucleases. In addition, the reported method is limited in performance to normal or specific enzymes such as exonucleases.

Fluorescent detection methods are one of the most sensitive detection techniques in use today. Enzymatic molecules of the Chamtomole ( ^10-21 M) content were analyzed using fluorescent microanalysis. Single enzyme molecules were detected with oil dispersed droplets by fluorescence microscopy. Each enzyme molecule was treated by electrophoresis using capillary tubes and monitored by fluorescence spectroscopy [Xue, Q., Yeung, ES, Differences in Chemical Reactivity of Individual Molecules of an Enzyme , Nature , 1995, 373 , 681-683]. Detection assays for coliform bacteria using β-galactosidase as marker enzyme have been developed. Fluorescence was found to be 250 times higher in sensitivity and reduced in detection time by 5 hours [Van Poucke, SO, Neils, HJ, Development of a Sensitive Chemiluminometric Assays for the Detection of β-Galatosidase in Permeabilized Coliform Bacteria and Comparison with Fluorometry and Colorimetry, Appl. Env. Microbiol. , 1995, 61 , 4505-4509.

The present invention provides a reagent comprising at least one enzyme specific cleavable polynucleotide substrate, which schematically retains at least one fluorescent site in close proximity to at least one other adjacent fluorescent site, wherein the adjacent fluorescent sites fluoresce each other. The fluorescent part can be easily detected by fluorescence measurement technique when the substrate is cut.

The reagents include enzyme specific cleavable polynucleotide substrates containing self-complementary single-stranded polynucleotides each having a fluorescent moiety at the 3 'end and the 5' end; A polynucleotide substrate comprising two complementary hybridized polynucleotide chains each containing a 3 'end and a 5' end having a fluorescent moiety at each end of one or both 3 '/ 5' pair (s); Or a polynucleotide substrate containing single-stranded polynucleotides having two or more pendant adjacent fluorescent portions along the length direction.

In addition, the present invention

(1) a reagent comprising at least one enzyme specific cleavable polynucleotide substrate which retains at least one fluorescent portion in close proximity to at least one other adjacent fluorescent portion, wherein the adjacent fluorescent portions are quenched from each other but separated A reagent that can be easily detected by fluorescence measurement techniques, and (2) a test sample that may contain the specific enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed is determined by cleavage of the polynucleotide, separation of the fluorescent moiety, fluorescence Provided is a biological assay comprising the step of formulating a test sample that will lead to an increase in strength.

As another embodiment of this assay, the method may further comprise measuring an increase in fluorescence intensity.

Fluorescence can be readily observed by conventional techniques such as radiation energy irradiation, fluorescence microscopy, 96 well plate reader or flow cytometer.

The present invention also provides a method of preparing an enzyme specific cleavable polynucleotide reagent having a quenched fluorescent moiety. This method involves preparing an enzyme specific cleavable polynucleotide containing reagent that quenches each other through dye dimerization but retains a fluorescent moiety that can be detected by fluorescence measurement techniques when separated. This method comprises at least one fluorescent compound having at least one fluorescent moiety and at least one reactive group,

(1) a self-complementary single-stranded polynucleotide having at least one end group reactive with at least one of the above-mentioned fluorescent compounds, and (2) at least two portions having a suspending group reactive with at least one of the above-mentioned fluorescent compounds, other than the terminal in the polynucleotide. Single-stranded polynucleotide having a position of and (3) one or more reactive end groups reacting with at least one of the above fluorescent compounds, 3 'and / or 5 on one or both of the 3' / 5 'pair (s) 'Combining enzyme specific cleavable polynucleotides selected from the group consisting of complementary polynucleotides each retained at each end of the chain, and under reactive conditions to produce the reagents.

In this specification,

"Dye dimerization" means the formation of a non-covalent complex between two dye groups,

"Dye stacking" means the formation of a non-covalent complex between two or more dye groups, wherein the two complexed dye groups are called "dimers" and the three complexed dye groups are "trimers" It is called.

By "enzyme specific cleavable polynucleotide" is meant a linear sequence of nucleotides containing a specific bond that is susceptible to cleavage by a specific enzyme.

The term "fluorescence" means that the material emits light of a certain wavelength when absorbing light of different wavelengths, where the emission of light occurs only during light absorption.

"Fluorescent quantum yield" means the ratio of the number of fluorescent photons emitted by a light emitting material to the total number of photons absorbed by the material.

The term "fluorescent substrate" refers to a material that is substantially non-fluorescent to produce fluorescent compounds upon enzymatic action.

The term “fluorophore”, “dye portion” or “dye group” means a molecule or portion of a molecule that emits light of a predetermined wavelength when it is stimulated by absorbing light of different wavelengths (usually short wavelength).

The present invention provides advantages over conventional assays that have been used for the detection and identification of microorganisms. The present invention provides a rapid and convenient method for detecting the presence and / or measuring the activity of intracellular and extracellular enzymes using enzyme specific cleavable polynucleotide substrates of fluorescent moiety retention, also referred to herein as fluorogenic nuclease substrates. Provide a method. This method can also be adapted to sequence dependent tests or sequence independent tests. The methods and reagents of the present invention have resulted in improved accuracy, faster detection and lower total cost in detecting and identifying microorganisms. In various embodiments, the present invention takes advantage of measurable fluorescence differences that allow for nuclease detection. As a preferred embodiment, the present invention exhibits a higher signal intensity upon cleavage compared to the much lower signal intensity upon quenching, and preferably a fluorescence that acts in the visible range to minimize interference by basic fluorescence of the propagation medium. Provide originality indicators.

The present invention specifically shifts the emission wavelength from the far ultraviolet region (about 350-400 nm) to the 500-600 nm region of the electromagnetic spectrum and reduces the effect of the fundamental signal intensity of the original reagent to detect the presence of nucleases. This provides a more accurate and sensitive measurement method. Another advantage of the present invention is that it is a homogeneous biological assay that does not require a colorant. Thus, it does not require time-consuming separation steps such as in enzyme-linked immunosorbent assays (ELISA) and assay formats such as bioluminescence. Detection and identification of microorganisms can be performed using a basic identification medium.

As will be apparent from the present specification, the assays of the present invention are characterized by more efficient use of reagents, labor, time and devices. In addition, the careful design and selection of polynucleotides and fluorophores attached thereto offer the possibility of simultaneously investigating several bacteria and / or enzymes. The analytical methods described herein are closely related to the detection and identification of microorganisms, sterility confirmation, pharmaceutical drug discovery, clinical analysis, nucleic acid hybridization, sequencing and amplification.

1 is a schematic diagram illustrating the conceptual mechanism by which a fluorogenic nuclease substrate is cleaved by a bacterium producing enzyme, wherein the fluorogenic nuclease substrate shows intramolecular dimerization, wherein the 3 'end of the polynucleotide and 5 The self-complementary single-stranded polynucleotide having a fluorescent moiety attached to the terminal is quenchable, and when the polynucleotide is cleaved, the fluorescent moiety can be separated and easily detected.

FIG. 2 is a schematic diagram illustrating the conceptual mechanism by which the fluorogenic nuclease substrate is cleaved by a bacterial producing enzyme, wherein the fluorogenic nuclease substrate is located in close proximity to each other through the dye stack and is suspended from each other. Retaining single-stranded polynucleotide, when the polynucleotide is cleaved, the fluorescent part is separated and can be easily detected.

FIG. 3 is a schematic diagram illustrating the conceptual mechanism by which a fluorogenic nuclease substrate is cleaved by a bacterial producing enzyme, wherein the fluorogenic nuclease substrate shows intermolecular dimerization, and the 3 'end of two complementary polynucleotides And two complementary hybridized polynucleotide chains having two fluorescent portions labeled at each of the 5 'and 5' ends respectively quenched, and when the polynucleotide is cleaved, the fluorescent portions can be separated and easily detected.

FIG. 4 shows a tetramethylhodamine-labeled 25-mer double-strand, which appears before (line b) and after incubation (line a) of micrococcus nuclease at 37 mmol in 100 mmol bicarbonate buffer (pH 8.9). It is a graph depicting the relative fluorescence intensity of polynucleotides.

FIG. 5 shows tetramethylhodamine-labeled 24-mer self-complementarity which appears before (line c) and after incubation (line d) of micrococcus nuclease at 100 mmol bicarbonate buffer (pH 8.9) at 37 ° C. It is a graph which shows the absorption spectrum of single stranded polynucleotide.

details

The present invention discloses a new method for providing a reagent containing a nuclease substrate. The substrate separates the quenched fluorescence upon exposure to the nuclease to be analyzed, thereby detectably increasing fluorescence.

This nuclease substrate is prepared by first specifying the polynucleotide to be used. This is done by selecting a base or base pair (A / T or G / C) to be included in the polynucleotide. The arrangement may be an arrangement in which the fluorescent portions are arranged close together. Hybridization can be used to obtain a suitable arrangement. Hybridization is formed when the complementary base pairs of polynucleotides are attracted to each other. Possible arrangements include, for example, self-hybridization, ie, self-complementary single-stranded polynucleotides in which fluorescent dye moieties are attached to both overlapping 3 'and 5' ends in order to bring the fluorescent parts attached to each end of the polynucleotide into proximity to each other. have. In another arrangement, either one of the 3 '/ 5' pairs has a fluorescent moiety at each end of both sides, and the polynucleotide hybridizes to form a double-stranded polynucleotide so that one end of each single-stranded polynucleotide is Fluorescent moieties attached to both ends include two mutually complementary single-stranded polynucleotides each having a 3 'end and a 5' end, which are in close proximity to each other. Another method involves a single-stranded polynucleotide in which two or more fluorescent dye moieties are suspended from the polynucleotide chain and are spaced very closely along the chain at appropriate lengths so that the fluorescent moieties are quite close to each other, resulting in quenching of fluorescence. Include.

In the case of the hybridized form, the degree of hybridization may be sufficient to cause the attached fluorescent parts to be in close contact with each other. In general, approximately 5 base pairs out of the base pairs closest to the 3 '/ 5' ends to which the fluorescent part is attached are sufficient to hybridize. This hybridization number may vary depending on factors such as the type of base pair in the polynucleotide and the dimerization constant of the fluorescent part.

In addition, the polynucleotide is specified to include one or more bonds that can be cleaved by the nuclease to be analyzed. Strategically, these bond (s) are placed in the polynucleotide such that the fluorescence is separated upon cleavage. For self-complementary single-stranded polynucleotides or for two mutually complementary polynucleotides forming a double-stranded polynucleotide, the cleavable bond (s) should generally be located at or near the end of the polynucleotide to which the fluorophore is attached. In the case of single-stranded polynucleotides having a suspended moiety, cleavable bonds should generally be located between the dye moieties.

In addition, the binding site for the selected fluorescent moiety is introduced into the polynucleotide at the specific position where the fluorescent moiety is desired. Such sites may be located at the ends (ends) of the polynucleotides as in the case of hybridized polynucleotides, or along the polynucleotide chain, ie the backbone, as in the case of single-stranded polynucleotides to which a suspension dye is attached. . These binding sites include reactive groups such as aliphatic primary amines that can readily react with fluorescent compounds, including fluorescent moieties to be attached to the polynucleotide.

Once the polynucleotide is specified, the polynucleotide can be synthesized, for example, by known manual or automated DNA synthesis methods. Thereafter, the polynucleotide is reacted with at least one compound containing a fluorescent moiety. Fluorescent compounds generally include reactive groups that react with reactive groups on the polynucleotides. The reaction is carried out under concentration conditions, stirring conditions, pH conditions, ionic conditions and temperature conditions suitable for attaching the fluorescent moiety to the polynucleotide at predetermined positions. Specific requirements depend on the fluorescent compound used and the reactive groups present in the polynucleotide, respectively. pH is adjusted using a buffer such as carbonate buffer or bicarbonate buffer. The fluorescent portion used in the present invention has dimerization or stacking ability, so that fluorescence may be quenched.

In addition, the present invention

(1) a reagent comprising at least one enzyme specific cleavable polynucleotide substrate which retains at least one fluorescent portion in close proximity to at least one other adjacent fluorescent portion, wherein the adjacent fluorescent portions are quenched from each other but separated A reagent that can be easily detected by fluorescence measurement techniques, and (2) a sample that may contain a specific enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed is determined by cleavage of polynucleotides, separation of fluorescence, and fluorescence intensity. It provides a biological assay comprising the step of formulating a test sample that will lead to an increase in.

As another embodiment of this assay, the method may further comprise measuring an increase in fluorescence intensity. This can be done with radiation energy irradiation, fluorescence microscopy, 96 well plate reader or flow cytometer.

1 illustrates a preferred embodiment of the present invention. Polynucleotide 10 may be synthesized to be self-complementary, that is, to self-hybridize in the chain. When the fluorescent dye portions 11 and 12 are attached to the 3 'end and the 5' end, the dye portions are sufficiently in close contact and quenched mutually, so that the fluorescence is very weakly observed. When treated with nucleases 13 produced by bacteria 14, the polynucleotide 10 is cleaved at sites such as (15), (16) and (17) to destroy the self-hybridized structure, A fragment 18 and non-dimeric dye portions 11 'and 12' are produced, which dye freely fluoresces upon dissociation. In this embodiment, the length of the polynucleotide chain should be of sufficient length so that the polynucleotides overlap to bring the fluorescent dye moiety close. In general, approximately 15 base pairs are sufficient.

2 shows a second preferred embodiment of the invention. The plurality of fluorescent dye portions 22 are attached to the polynucleotide 20 including various sites 24 that are susceptible to enzymatic cleavage. The dye portions 22 are disposed close enough to each other so that the fluorescence of the dye portion 22 is quenched. The conjugate (including the polynucleotide 20 and the fluorescent dye portion 22) is treated with the nuclease 26 produced by the bacteria 28 to cleave the polynucleotide 20. Cleavage produces nucleotide fragments 29 and separates the dye moieties 22 'from each other in the medium, allowing them to fluoresce freely and be easily detected. In this embodiment, the fluorescent portions 22 are preferably separated by less than 10 nm between bases along the backbone of the polynucleotide. This length is determined by an indirect method based on the known or estimated length of the polynucleotide base. The arrangement shown in FIG. 2 has a series of pairs of typed or grouped fluorescent moieties and can be used to test multiple types of bacteria or enzymes, where each type can be distinguished from each other using fluorescence measurement techniques. Each portion in each pair or group may be bound by cleavable bonds that are susceptible to cleavage by other bacterial enzymes or may be located on opposite sides of the cleavable bonds.

Figure 3 shows another preferred embodiment of the present invention. The complementary hybridized polynucleotides 30 and 31 have fluorescent dye portions 32 and 33 attached to their 3 'and 5' ends, respectively. The nuclease 36 produced by the bacterium 38 cleaves the polynucleotide (s) 30 and / or (31) at the cleavage site 34 to dissociate the nucleotide fragment 39 with the nondimerized dye moiety. Produces 32 'and 33', which are easily detectable. Still other embodiments may include dye moieties attached to each 3 'end and 5' end of each complementary polynucleotide. The arrangement shown in FIG. 3 can be used to test two kinds of bacteria or enzymes simultaneously. The method labels each complementary 3 '/ 5' end pair with a different kind of fluorescent moiety that can be distinguished from each other using fluorescence measurement techniques, and by a different kind of bacterial enzyme near each end of the double-stranded polynucleotide. A double-stranded polynucleotide is constructed so that a cleavage site that is easy to cleave is located.

Fluorescent substrates are molecules that vary from substantially nonfluorescent to highly fluorescent by enzymatic hydrolysis. This substrate is widely used as a molecular probe in the research and testing of viral and bacterial enzymes such as proteases, nucleases, saccharases, phosphatase and kinases. Fluorescence of the substrate can be readily observed by conventional techniques such as radiation energy irradiation, fluorescence microscopy, 96 well plate reader or flow cytometer.

Enzyme-specific cleavable polynucleotide substrates having fluorescent moieties or fluorogenic nuclease substrates useful in the present invention can be prepared by chemical reaction of polynucleotides with fluorescent dyes such as fluorescein and rhodamine to dimerize or stack. Can be. Useful polynucleotides are commercially available (eg, GeneMed Biotechnologies, South San Francisco, Calif.). In order to prepare the reagents of the present invention, two or more fluorescent moieties cause dimerization or stacking (eg, through substantially hybridization, proximity, interchain hybridization, intrachain hybridization, etc.) to mutually extinguish the fluorescence of the fluorescent moiety. (If the fluorescent part is the same, it is called "self quenching.") The said fluorescent part is couple | bonded with a polynucleotide.

Fluorescent dye quenching can occur through many known mechanisms, including energy transfer and dye dimerization. In this case, when energy is input and generally irradiates light of a specific wavelength to excite a fluorescent dye molecule or part, energy is transferred from the dye part to another part rather than dissipated by fluorescence. Energy transfer (also called the posterior dipole interaction) occurs between the fluorescent donor and the receptor, whether or not fluorescent. Energy transfer generally occurs when the distance between the donor and acceptor on the substrate backbone is about 10 nm. Through energy transfer, the fluorescence of the donor section is quenched. On the other hand, dye dimerization or dye stacking occurs when two or more fluorescent groups are brought close enough to each other to form stable complexes such as dimers and trimers through the interaction of their planar aromatic rings. Each fluorescent part acts on the other fluorescent part (s) to quench each other. The absorbance spectrum of the dye in the dimer state or the stacked state is substantially different from that of the same dye in the energy transfer pair. The dye dimer absorbance spectrum shows a characteristic decrease in absorbance of the absorbance main peak as the dye concentration increases, while a characteristic increase in absorbance of the shoulder peak. This phenomenon is illustrated in FIG. 5. For the dye molecules shown in the graph of FIG. 5, the absorbance main peak appears at about 560 nm and the shoulder peak at about 530 nm. The dimeric sample (c) exhibits an absorbance peak with an absorbance unit (AU) of about 0.03 at both the absorbance main peak and the shoulder peak, whereas the nondimeric sample (d) has a main absorbance peak of about 0.06 AU and about 0.028 AU. Leveled shoulder peaks are shown. The phenomenon appearing in the dimeric sample as such is usually referred to as "band segmentation". Dimerization or lamination occurs through the formation of steady state complexes (ie, through physical close proximity), while energy transfer interactions occur through space. Thus, characteristic spectral changes in dye dimerization are not observed in dyes that interact by energy transfer mechanisms.

The quenching mechanism of dimerized or stacked dye pairs or groups of dyes used in the present invention is different from the quenching mechanism of fluorescent energy transfer donors and acceptors. Examples of dyes that exhibit dimerization or stacking properties when binding to polynucleotides in close proximity to each other include homodimers or when in solution at sufficiently high dye concentrations (e.g., 10 ^-2 to 10 ^-4 M). It generally includes dyes having a planar aromatic structure so as to form a heterodimer. This phenomenon is clearly undesirable in many fields, but is advantageously used in the present invention. The concentration may be increased by increasing the amount of dye in a unit dose or by placing two (or more) dye portions physically in close proximity together, such as on polynucleotides or other small molecules. When the dye portion is disposed in close proximity, the local concentration is effectively increased, and the polynucleotide carrying the fluorescent portion is in the quenched state regardless of the total concentration.

Planar aromatic dyes belonging to the fluorescein and rhodamine series, fluorescein, tetramethyltamine (TMR), rhodamine B, X-rhodamine and the like are representative dyes which dimerize when the concentration is sufficiently high. Other suitable fluorescent dyes include, for example, cyanine, boron-heterocyclic dyes, such as 4,4-difluoro-4-boreta-3a-azonia-4a-aza- s -indacene (in Eugene, Oregon, USA). Molecular Probes, Inc. under the tradename BODIPY ™). Rhodamine-based dyes such as tetramethyltamine, X-rhodamine and rhodamine B exhibit very high fluorescence quantum yields (about 0.85) in the visible wavelength range (400-700 nm). Therefore, this dye is often used to detect trace amounts of material.

The dye portion used in the present invention may further include a reactive end group for easily attaching the fluorescent portion to the polynucleotide. This terminal group may include succimidyl ester, maleimide, ester, thioisocyanate, isocyanate and iodoacetamide. Dye molecules with such reactive groups already attached are commercially available, for example, from Molecular Probes, Inc. (Eugene, Oregon, USA).

Polynucleotides used in the present invention must have at least one enzyme specific cleavable site. Fluorescent quenched polynucleotides having two or more fluorescent moieties will increase the fluorescence intensity by cleaving the dimerized fluorescent moiety or stacked fluorescent moieties upon cleavage. As a result, the fluorescence is easily detected, which suggests the presence of specific enzymes and, where applied, the presence of bacteria producing enzymes. Due to the interaction between the transition dipoles of the resonant dimer structure or the laminated structure, the fluorescence quantum yield of the dimeric structure or the laminated structure is significantly lower when the polynucleotide is intact. However, if the polynucleotide is enzymatically cleaved to dissociate the dimer or lamination structure, the fluorescent portion can be separated, resulting in a significantly higher fluorescence quantum yield in aqueous solution due to the inherent high fluorescence. In this way, the increase in fluorescence and the presence of hydrolase can be distinguished. On this basis, homogeneous enzyme assays can be devised which do not require a colorant and a separation step. This assay involves making an enzyme specific cleavable polynucleotide bearing a fluorescent site in solution and adding an enzyme analyte, where any enzyme present in the analyte cleaves the polynucleotide to dimerize Or reduces lamination and consequently increases fluorescence. Thus, enzyme activity is directly related to the net increase in fluorescence intensity. If the enzyme is secreted from active metabolic cells, fluorescence intensity can be associated with the metabolic activity of the cells.

The ability of one enzyme molecule to convert millions of reagent molecules is an amplification process. This amplification is further enhanced when enzymes are obtained from live cell culture because more enzyme molecules are produced as the cells proliferate. Using these two factors in conjunction with fluorescence techniques yields a preferred method for the rapid, sensitive and specific detection of bacteria.

Representative polynucleotides useful for generating fluorogenic nuclease substrates must retain the essential chemical bonds that are the target of the nuclease to be analyzed. For example, as described below, micrococcus nucleases are known to hydrolyze polynucleotides at phosphodiester bonds, so any polynucleotide having this feature can be a micrococcus substrate.

Chemical modification of polynucleotides is carried out using nucleotide analogs and DNA synthesis reagents. For example, phosphoramidites containing aliphatic primary amines are polynucleotides via automated DNA synthesizers such as the Nucleic Acid Synthesis and Purification System (Perkin Elmer Applied Biosystems, Foster City, Calif., Model # ABI 3948). May be introduced at the desired position in the chain. Terminal modifiers and amino modified deoxythymidines (dT) for use in introducing aliphatic amines having C ₃ , C ₆ or C ₉ linkages at the 3 ′ end and / or 5 ′ end of the polynucleotide are commercially available. The amine can then be reacted with various labels, such as biotin, fluorescent dyes or alkaline phosphatase, to form polynucleotide conjugates. Common uses of such modified polynucleotides include (i) non-radioactive hybridization probes, (ii) sequence specific cleavage of DNA, (iii) automated DNA sequencing and (iv) affinity chromatography. Other parts that may be added to the ends of the polynucleotide to react with the moiety of dye to be attached include those having reactive H groups such as free thiols (SH), carboxyl groups and OH groups. Preferred are amines and thiols.

Enzymatic cleavage is carried out by contacting a fluorogenic nuclease substrate, i.e., a reagent with a particular enzyme to be analyzed. Target enzymes suitable for use in the present invention include all enzymes generally classified as nucleases, ie enzymes that hydrolyze nucleotide bonds, for example thermonucleases or Staphylococcus nucleases.

If the enzyme is an intracellular enzyme, the enzyme can be prepared for easy contact with the fluorogenic nuclease substrate using an agent that increases the permeability of the outer membrane (OM) of the enzyme bearing bacteria. Ethylenediaminetetraacetic acid (EDTA), which affects the outer membrane barrier of Gram-negative intestinal bacteria, is generally used for this purpose. Under certain conditions, OM may rupture to release intracellular enzymes.

For rapid and sensitive tests, it is important to maximize the desired signal relative to the signal-to-noise ratio, ie in this case unwanted background signals such as the bacterial and proliferation media's own fluorescence. Most conventional fluorogenic substrates emit light in the far ultraviolet wavelength region (350-450 nm) in which most bacterial growth medium exhibits significant self fluorescence. For example, a general culture medium of Staphylococcus aureus shows two significant maximal releases at 425 nm and 475 nm when excited at 360 nm. To avoid this problem, substrate concentrations are generally used at high concentrations. However, this increases the cost of analysis, can be highly toxic to the organism, and sometimes precipitates the substrate. Accordingly, the present invention uses a fluorescent dye (e.g., tetramethyltamine: Ex = 550 nm, Em = 580 nm) emitted at a wavelength far longer than the background fluorescence wavelength to red shift the detection wavelength toward the visible light spectrum to self fluorescence. Overcome the disadvantages of interference In addition, the technical idea disclosed in the present invention may be applied to other dyes that shift the emission wavelength to a longer wavelength. In general, useful dyes have the following characteristics: high extinction coefficient (> 80,000 cm ⁻¹ M ⁻¹ ), high quantum yield (> 0.85 in aqueous solution), spectrum insensitive to solvents and pH, aqueous solubility, light Stability and preferably a dimerization constant of at least 10 ⁻³ .

Many Staphylococcus strains exhibit DNase activity. An enzyme that has traditionally been used for identification purposes is the extracellular DNase of S. aureus . S. aureus nucleases are secreted in large quantities, thermostable, and require Ca ²⁺ instead of Mg ^{2+ for their} activity. This feature is used to distinguish S. aureus from other Staphylococcus in detecting Staphylococcus contamination in food. In addition, extracellular DNase is also produced by Streptococcus (Streptococcus) species, Serratia marcescens (Serratia) and Bacillus species (Bacillus) species.

Micrococcus nuclease, an extracellular enzyme of S. aureus , is used as a model system to confirm the utility of the present invention. This enzyme cleaves the 5 'phosphodiester linkage of the polynucleotide chain, is stable at high temperatures (> 60 ° C), remains active and is commonly used as an indicator of S. aureus . The presence of nuclease activity at 60 ° C. is a confirmatory test for S. aureus . Micrococcus nucleases do not require cofactors for their function. This is a feature that can reduce the complexity and cost of analysis.

In summary, the present invention provides novel nuclease reagents, analytical methods using the reagents, and methods of making the reagents. This reagent includes single- or double-chain synthetic polynucleotides having two or more fluorescent moieties that may be dye molecules (eg, tetramethylhodamine). Fluorescence increase after nuclease hydrolysis and cleavage is on the order of 2-6 fold depending on the specific form of the reagent. This reagent is combined with the "double cascade" phenomenon, that is, each enzyme molecule can convert millions of reagent molecules and release more enzyme molecules as the bacterial cells divide and multiply exponentially. Fast, sensitive and specific bacterial detection methods will be provided. The present invention provides, but is not limited to, microbial detection and identification, sterilization confirmation, drug discovery, enzyme assays and immunoassays.

The objects and advantages of the present invention will be described in more detail through the following examples, but the specific materials and amounts and other conditions and details used in these examples should not be understood to unduly limit the present invention.

Test Methods

Fluorescence spectrum

All fluorescence measurements were taken with a fluorescence spectrometer commercially available under the trade name FLUOROLOG ™ from Specs Instruments, Edison, NJ. In a typical measurement, 3 ml of assay solution was placed in a quartz cuvette and placed in the sample fixture of the fluorometer. The fluorescence emission spectrum of the assay solution was measured at fixed excitation wavelengths at room temperature. The widths of the excitation and emission slits of the fluorometer were both set to 0.5 mm. Fluorescence spectra of the nuclease containing or non-containing assay solution were measured under the same instrument conditions.

Example 1

In this example, two tetramethyltamine (TMR) dye moieties were joined at the 3 'end and the 5' end of the self-complementary 24-mer polynucleotide. The dye portions were brought into close contact with each other by in-chain hybridization, and as a result, fluorescence was quenched. Enzymatic cleavage at multiple sites disrupted the aligned structure. As a result, the dye moiety is dedimerized to show a significant increase in fluorescence. Such a technical idea is schematically illustrated in FIG. 1.

Self-complementary polynucleotide NH ₂ CH ₂ CH ₂ CH ₂ -5'- AAC AAA GGA TAA TTA TCC TTT GTT -3'-CH ₂ CH ₂ CH ₂ NH ₂ is Ginmed Biotechnology, Inc. From San Francisco, CA, and the chemical structure was confirmed by DNA sequencing. About 270 nmole of this polynucleotide was dissolved in 600 µl of deionized water. 90 nmole (200 μl) of polynucleotide solution was added to tetramethyltamine (TMR) succimidyl ester (Molecular Probes, Inc., Eugene, Oregon, USA) 4 mg [100 mM sodium bicarbonate buffer predissolved in 1 drop of dimethylsulfoxide (DMSO) in (pH 8.3)] and at room temperature. This mixture was a sufficient volume (ie, about 2 mL) to maintain a proper pH. In this amidation process, the ester of the TMR moiety reacted with the amine moiety attached to the polynucleotide.

The reaction mixture was pipetted to a size exclusion column containing epichlorohydrin crosslinked (alkali conditions) dextran gel (commercially available under the tradename SEPHADEX ™ G25 from Pharmacia Biotech, Inc.) as a filler material. Labeled polynucleotides were isolated from unreacted dyes by loading into Pharmacia Biotech, Inc., Model PD-10, Piscataway, NJ). The reaction mixture was passed through the column under gravity. The resulting DNA fractions were further purified by high performance liquid chromatography (HPLC) on a C18 reversed phase column (Waters Corporation, Milford, Mass., Model 600E HPLC). As the mobile phase of the column, a gradient of 0.1 M triethylamine acetate (TEAA) and acetonitrile (ACN) in water was used, with the volume ratio of TEAA: ACN decreasing linearly to 8:92 over 30 minutes starting at 92: 8. I was. The final conjugate was the polynucleotide structure with TMR addition on each of the aliphatic amine linkages at the 3 'and 5' ends.

HPLC fractions containing the purified conjugate were pooled, frozen with dry ice and then vacuum dried at room temperature. The anhydrous conjugate was then dissolved in 5 ml of 100 mM sodium carbonate buffer (pH 8.9). As shown in FIG. 5, an absorption spectrum measured using a spectrophotometer (Shimazu Scientific Instruments, Model MPC-3100, Columbia, MD) shows the characteristic band splitting characteristics of dimerized tetramethyltamine. Indicated. A small amount (about 0.5 mg) of Micrococcus nuclease (obtained from Sigma Chemical Company, St. Louis, MO) was added to 3 ml of the buffer solution. This mixture was incubated overnight at 37 ° C. for enzymatic hydrolysis of the 5′-phosphodiester bond of the polynucleotide bearing fluorescent moiety. This solution was then cooled to room temperature. The fluorescence spectra were then measured at room temperature using a FLUOROLOG ™ fluorescence spectrometer. As a result of the enzyme cleavage, the absorbance at 560 nm increased from 0.03 AU to 0.06 AU at 560 nm, and the fluorescence intensity increased from 12000 to 28000 at 580 nm.

In another test, the length of the aliphatic amine linkage at the 5 'end was increased to make the deposition more efficient between the two dye sections. The following polynucleotides with longer aliphatic amine linkages were obtained from Gin Med and were labeled and tested under conditions similar to those described above.

H ₂ N- (CH ₂ CH ₂ O) _6- (CH ₂ ) ₃ -5'- AAC AAA GGA TTA TAA TCC TTT GTT -3'-CH ₂ CH ₂ CH ₂ -NH ₂

H ₂ N- (CH ₂ CH ₂ O) _9- (CH ₂ ) ₃ -5'- AAC AAA GGA TTA TAA TCC TTT GTT -3'-CH ₂ CH ₂ CH ₂ -NH ₂

A two-fold increase in fluorescence was observed with each test.

Example 2

In this example, the suspended tetramethylhodamine dye portion was attached to a plurality of positions in the polynucleotide sequence. Due to the proximity, the dye portions were laminated together to quench fluorescence. Enzymatic cleavage of the polynucleotide at multiple positions disrupted the conjugate, dissociating the dye portion into a single portion from the dimer or stacked state, while simultaneously increasing the fluorescence intensity. This technical idea is schematically illustrated in FIG. 2.

The polynucleotide chain was designed to include a total of four amino groups in the replacement base as follows. 5'-TTT TTT Z T Z T Z T Z TTT TTT-3 '. Wherein Z is an amine modified C ₆ derivative of deoxythymidine (dT) (Glen Research, Sterling, Va., Cat. No. 10-1039-90). Synthesis of polynucleotide chains was synthesized by Ginmed Biotechnology, Inc. About 45 nmole of polynucleotide was dissolved in deionized water and 1 mg of tetramethyltamine (TMR) succimidyl ester [preliminarily dissolved in 1 drop of dimethyl sulfoxide (DMSO) in 100 mM sodium bicarbonate buffer (pH 8.3)]. And reacted at room temperature. The conjugate was purified under the same conditions as in Example 1. Enzymatic hydrolysis was performed by reacting overnight at 37 ° C. using a small amount (about 0.5 mg) of micrococcus nuclease in 100 mM sodium bicarbonate buffer (pH 8.9). The fluorescence spectrum was measured at room temperature with an excitation wavelength of 520 nm, and the fluorescence intensity at 575 nm increased about 2 times after enzymatic hydrolysis.

Example 3 (Comparative Example)

In this example, quenching (quenching of one dye portion of the dye portions) was carried out by energy transfer, not dye dimerization.

DNase activity was analyzed by fluorescence energy transfer using the following two polynucleotides obtained from Ginmed Biotechnology, Inc.

A. 5 'TTA X TA A Y T CCG TTT AA 3'

B. 5 'TTA Z TA A Y T CC X TTT AA 3'

Wherein X is tetrachlorofluorescein (receptor), Y is fluorescein (donor), Z is another receptor suspended from the amino modified C ₆ [ Z of deoxythymidine (dT), tetramethyltamine (TMR) attachment. Two different dyes, the donor and the receptor, X and Y, in the A configuration were inserted into the polynucleotide sequence. Unlike Example 2, in which all dye moieties are suspended from polynucleotides, in this example, the donor and one receptor were bottled into the polynucleotide backbone itself. The B configuration is similar to A except that the second receptor (TMR) is suspended from Z. When the polynucleotide was intact, the donor and the receptor were located within energy transfer distance of each other. The excitation state energy of fluorescein was non-radioactively transferred to tetrachlorofluorescein (and / or tetramethyltamine in B) to quench donor fluorescence. Receptor fluorescence is not quenched. After DNA is enzymatically hydrolyzed, the donor and receptor are separated, resulting in little energy transfer. After hydrolysis by micrococcus nuclease, the donor fluorescence of Compound A increased about 30% at about 530 nm upon excitation at 490 nm. Upon visual observation, the fluorescence changed from orange blue to dark blue, indicating that the energy transfer efficiency was reduced. Similar results were observed for compound B.

Example 4

In this example, tetramethyltamine was attached to aliphatic amine linkages present at the 3 'end and the 5' end of the two complementary polynucleotides. The dye portions were brought close to each other through interchain hybridization and fluorescence was quenched. By enzymatic cleavage at multiple sites along the double-stranded polynucleotide, disrupting the ordered structure, the dye portion is dissociated from the dimer to the monomer to obtain a significant increase in fluorescence. This technical idea is schematically illustrated in FIG. 3.

Two complementary 25-mer polynucleotides were synthesized by Ginmed Biotechnology, Inc.

NH _2- (CH ₂ ) ₃ -5'- AAC AAA GGA TTA TAG TGG GAA ATC A -3 '

NH _2- (CH ₂ ) ₃ -3'- TTG TTT CCT AAT ATC ACC CTT TAG T -5 '

About 22.5 nmole of each polynucleotide was reacted with 1 mg of tetramethyltamine (TMR) isothiocyanate in 100 mM sodium carbonate buffer (pH 9.0), respectively, overnight at 23 ° C. under stirring (shaking). The dye was first dissolved in 1 drop of DMSO and then mixed with the polynucleotide. A thio-urea bond was formed between the amine group attached to the polynucleotide and the isothiocyanate of the TMR moiety. Each reaction mixture was passed through a size exclusion column described in Example 1 (Pharmacia Biotech, Inc., Piscataway, NJ, PD-10) to separate labeled species from unreacted dye molecules. . Melting point T _m was calculated based on the ratio of the ionic strength of the hybridization solution (Promega Corp., Madison, Cat. No. F123A, Catalog No. F123A) to the ratio of the GC pair content in the polynucleotide chain using the following equation.

T _m  = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% GC)-(600 / N)

Where N is an integer representing the number of bases in the polynucleotide chain. Through this equation, one can predict whether T _m is suitable for polynucleotides having a length between 14 and 60 to 70 nucleotides. The Na ⁺ concentration of the promega hybridization solution is 0.39. Based on the above formula, the T _m of the polynucleotide was calculated to be 63.8 ° C. Hybrids formed between short polynucleotides at a temperature 5-10 ° C. below T _{m were} likely to dissociate reversibly. Therefore, the reaction temperature should be at least 10 ° C. lower than T _m . In addition, in order to produce stable hybrids, hybridization with short polynucleotides should be performed under stringent conditions using high concentrations (0.1-1.0 picomoles / ml) of polynucleotides. In this example, hybridization of two chains with fluorescent dyes was performed overnight at 45 ° C. (19 ° C. lower than T _m ) in a highly stringent hybridization solution (Promega solution). The conjugate solution was observed to exhibit fluorescence intensity at 575 nm (Ex = 550 nm), which was observed at 37 ° C. in 100 mM bicarbonate buffer (pH 8.9) than before treatment with nuclease (MO) After an overnight incubation with the Sigma Chemical Company, St. Louis, Cat. This is shown in FIG.

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Claims (20)

A reagent comprising at least one enzyme specific cleavable polynucleotide substrate that retains at least one fluorescent portion in close proximity to at least one other adjacent fluorescent portion, wherein the adjacent fluorescent portions fluoresce each other by dye dimerization or stacking. Quenching, wherein the fluorescent part is easily detectable by fluorescence measurement techniques when the substrate is cleaved. delete delete delete The reagent of claim 1, wherein the polynucleotide substrate comprises self-complementary single-stranded polynucleotides having a 3 'end and a 5' end, each having a fluorescent moiety. The polynucleotide substrate of claim 1, wherein the polynucleotide substrate comprises two complementary hybridizing polynucleotide chains each having a 3 'end and a 5' end, each of which is one or both of the 3 '/ 5' pair (s). The terminal having a fluorescent moiety. The reagent of claim 1, wherein the polynucleotide substrate comprises a single stranded polynucleotide having two or more suspended contiguous fluorescent portions along its length. delete delete delete delete delete delete (1) a reagent comprising at least one enzyme specific cleavable polynucleotide substrate which retains at least one fluorescent portion in close proximity to at least one other adjacent fluorescent portion, wherein the adjacent fluorescent portions quench each other but are separated A reagent that can be readily detected by fluorescence measurement techniques, and (2) A test sample which may contain a specific enzyme to be analyzed, wherein the presence of the specific enzyme to be analyzed leads to cleavage of polynucleotides, separation of fluorescence, and increase in fluorescence intensity. Biological analysis method comprising the step of combining. delete delete delete A method for preparing a reagent comprising an enzyme specific cleavable polynucleotide having a fluorescent moiety detectable by fluorescence measurement techniques when quenched and separated from each other through dye dimerization or lamination, comprising: at least one fluorescent moiety and at least one At least one fluorescent compound having a reactive group, (1) a self-complementary single-stranded polynucleotide having at least one end group reactive with at least one of the above-mentioned fluorescent compounds, and (2) at least two portions having a suspending group reactive with at least one of the above-mentioned fluorescent compounds, other than the terminal in the polynucleotide. Single-stranded polynucleotide having a position of and (3) one or more reactive end groups reacting with at least one of the above fluorescent compounds, 3 'and / or 5 on one or both of the 3' / 5 'pair (s) 'Combining enzyme specific cleavable polynucleotides selected from the group consisting of complementary polynucleotides each retained at each end of the chain, and under reactive conditions for producing said reagents. delete delete

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