CN113897418B - Probe for detecting DNA point mutation, kit and application - Google Patents

Abstract

The invention relates to a probe, a kit and an application for detecting DNA point mutations. The probe has an AP site for being cleaved by endonuclease IV (Endo IV), and the AP site divides the probe into a first sequence and a second sequence; The mutant strand and the wild strand complement each other to form a first substrate and a second substrate, and the first substrate and the second substrate respectively have the first activity and the activity of being cleaved by endonuclease IV to form a free single strand with the second sequence. The second activity, the first activity is greater than the second activity. Since Endo IV has unique enzymatic activity on the truncated dsDNA structure of different first substrates and second substrates, a unique Endo IV probe is designed to form this special dsDNA structure with the target strand, The sequence dependence of Endo IV is greatly reduced, the universality of detection is broadened, and the cost of detection is reduced.

Description

Probes, kits and applications for detecting DNA point mutations

technical field

The invention relates to the technical field of single nucleotide mutation detection, in particular to probes, kits and applications for detecting DNA point mutations.

Background technique

Single nucleotide variation (SNV) or other types of various DNA point mutations are associated with a variety of human diseases, and various DNA point mutations have been recognized as biomarkers for clinical diagnosis and treatment of human cancer, such as TP53, EGFR and RAS gene mutations. However, mutant DNA (MT) often coexists with large amounts of wild-type DNA (WT) in clinical samples at low abundance levels (<0.1%). Currently, various methods for detecting DNA point mutations have been developed, including DNA sequencing, polymerase chain reaction (PCR), and nucleic acid hybridization. DNA sequencing (Sanger sequencing, next-generation sequencing, etc.) and PCR (allele-specific PCR, blocking PCR, droplet digital PCR, etc.) are the most commonly used strategies. However, both strategies require long reaction and detection times, high costs, and complicated optimization of experimental conditions, which put forward higher requirements for instruments and operators.

Nucleic acid probe hybridization-based strategies have attracted much attention due to their convenient and flexible strategy design and the lack of expensive equipment. However, the common problem of the probe-based DNA point mutation detection strategy is that the probe needs to be repeatedly optimized for experimental conditions; especially when detecting different SNVs, in order to determine the sensitivity and selectivity of the probe, it is often necessary to pass Optimization of reaction temperature, introduction of auxiliary strands and other methods to determine, which undoubtedly increases the cost of probe design.

Contents of the invention

In view of this, the object of the present invention is to provide a simplified probe which reduces the design cost.

In the first aspect, the embodiment of the present invention discloses a probe for detecting DNA point mutations, a probe for distinguishing DNA mutant strands and DNA wild strands, the probe is a single nucleotide strand, and the probe has an AP site for being cleaved by endonuclease IV, and the AP site divides the probe into a first sequence and a second sequence; the probe can be separated from the mutant strand and the The wild strand is complementary to form a first substrate and a second substrate, the second substrate has a mismatched base site corresponding to the mutation site of the mutant chain The first substrate and the second substrate have respectively the first activity and the second activity of being cut by endonuclease IV to form a free single chain with the second sequence, and the first activity is greater than the second activity.

In an embodiment of the present invention, the first substrate has a first double-stranded sequence, the mutation site of the mutant chain is in the first double-stranded sequence, and the second substrate has a second double-stranded sequence , the mismatched base position is in the second double-stranded sequence; the first double-stranded sequence and the second double-stranded sequence are correspondingly in the same sequence position. Wherein the same sequence position is the same in the sequence position of the dsDNA structure in the first substrate and the second substrate, and the probe can be formed with the partial sequence of the truncated target strand or the longer target strand as shown in Figure 4 dsDNA structure.

In the embodiment of the present invention, in the first double-stranded sequence, the 5' end of the mutant strand used to form the first double-stranded sequence is at a distance from the AP site 5' of the mutant strand Between 1 base upstream in the direction and 7 bases downstream in the 3' direction. Thus, the first substrate formed with the first double-stranded sequence has a higher activity of being cleaved by endonuclease IV.

In the embodiment of the present invention, in the first double-stranded sequence, the 5' end of the mutant strand used to form the first double-stranded sequence is located 3' away from the AP site of the mutant strand 1 base downstream in the direction. Thus, the first substrate formed with the first double-stranded sequence has the best activity of being cleaved by endonuclease IV.

In the embodiment of the present invention, in the first double-stranded sequence, the mutation site of the mutant chain is located 1 to 4 bases upstream from the AP site in the 5' direction of the mutant chain or 1 base downstream in the 3' direction. The first substrate formed with the first double-stranded sequence has a higher activity of being cleaved by endonuclease IV than the second substrate.

In the second aspect, the embodiment of the present invention discloses a probe for detecting DNA point mutations, including the probe involved in the first aspect, connected to the first sequence of the probe in the 5' direction of the AP site An upstream fluorescent group and a fluorescent quenching group connected downstream in the 3' direction of the AP site on the second sequence of the probe. The probe thus formed can be excited to generate fluorescence to characterize the enzyme cleavage activity of the substrate it forms.

In a third aspect, the embodiment of the present invention discloses a probe for detecting DNA point mutations, including the probe described in the first aspect, wherein the second sequence of the probe is a deoxyribozyme sequence.

In the fourth aspect, the embodiment of the present invention discloses a kit or reagent combination for detecting DNA point mutations, including the probe and endonuclease IV as mentioned in the first aspect.

In the fifth aspect, the embodiment of the present invention discloses a kit or reagent combination for DNA point mutation detection, including the probe involved in the first aspect, endonuclease IV, nanoparticles and auxiliary metal ions, and the nanoparticles are connected to There is a substrate labeled with a fluorophore and used for cleavage by the deoxyribozyme.

In the sixth aspect, the embodiments of the present invention disclose the probes involved in the first aspect, the second aspect or the third aspect, and the kits or combinations of reagents involved in the fourth aspect or the fifth aspect in the detection of single nucleotide mutants in the application.

Compared with the prior art, the present invention has at least the following beneficial effects:

The present invention designs a unique Endo IV probe by exploring the cutting activity of Endo IV to different dsDNA structures, so that it forms this special dsDNA structure, which greatly reduces the sequence dependence of Endo IV; and further combines DNAzyme and nano gold technology, which eliminates the need to modify the probe sequence with fluorescent and quencher groups. For different mutation sites, only the part that matches the mutation site needs to be changed, and the DNAzyme sequence remains unchanged, which greatly reduces the cost of probe design and testing costs.

Description of drawings

Fig. 1 is a schematic diagram of the principle of using Endo IV enzyme digestion to detect point mutations in the prior art provided by the embodiment of the present invention.

Figure 2 is the cleavage results of Endo IV for different substrates (based on FP-1 probe and C1 sequence) provided by the examples of the present invention; Figure 2A is a schematic diagram of the cleavage of Endo IV; 1 Hybridization at different positions and the cleavage process of Endo IV; Figure 2C is the result of the fluorescence increase response rate of different FP-1/C1-(X) cleaved by Endo IV; where C1-T38 indicates that the Target chain and FP-1 chain are all complementary.

Fig. 3 is the result graph of the fluorescence increase response rate of Endo IV for different FP-2/C2-(X) cleavage provided by the embodiment of the present invention (based on FP-2 probe and C2 sequence); wherein C2-T38 represents the Target chain and The FP-2 strands are fully complementary.

Figure 4 is the cleavage effect of Endo IV enzymes on substrates formed by different wild strands and mutant strands provided by the examples of the present invention; Figure 4A is a scheme for hybridization of FP-1 with MT or WT; Figure 4B is based on C1-(+1) As MT, C1-(+1/-XM) was used as WT to calculate the different fluorescence growth rates and DF values formed by FP-1/MT and FP-1/WT; Figure 4C used C1-T38 as MT to calculate FP The fluorescence growth rate and DF value of different substrates formed by -1 and CM1-T38; Fig. 4D is the fluorescence intensity response of C1-(+1), 0 means the target chain is only C1-(+1/-1M), Fig. The right panel of 4D shows the intensities at low levels of mutations in fluorescence abundance.

Figure 5 is the characterization of the modification process of Sub-AuNPs provided by the embodiment of the present invention; Figure 5A is a TEM image; Figure 5B is a normalized UV-visible spectrum; Figure 5C is a hydration particle size diagram; Figure 5D is a Zeta potential result, Figure 5E Agarose gel electrophoresis image results of AuNPs and Sub-AuNPs.

Fig. 6 is a schematic diagram of SNV detection based on DNAzyme and AuNPs provided by the embodiment of the present invention.

Figure 7 is the results of SNV detection based on DNAzyme and AuNPs provided by the embodiment of the present invention; Figure 7A is an agarose gel electrophoresis image of different reactants, lane M: DNAmarker; lane 1: free DNAzyme; lane 2: DP- 1; Lane 3: DP-1/MT; Lane 4: DP-1/WT; Lane 5: DP-1/MT+Endo IV; Lane 6: DP-1/WT+Endo IV; Lane 7: DP-1 +Endo IV; Figure 7B is a one-step detection method of SNV based on Endo IV and DNAzyme; Figure 7C is a two-step detection method of SNV based on Endo IV and DNAzyme; Figure 7D is the fluorescence intensity response results of one-step detection mode to different components; Figure 7E is the fluorescence intensity response results of the two-step detection mode to different components; in Figure 7D and Figure 7E, curve a: DP-1+Endo IV+Sub-AuNPs curve; curve b: DP-1+Endo IV+Sub- AuNPs +Mn ²⁺ ; Curve C: DP-1+C1-(+1)+Endo IV+Sub-AuNPs+Mn ²⁺ ; Curve D: DP-1+C1-(+1/-1M)+Endo IV +Sub-AuNPs+Mn ²⁺ .

Figure 8 is a graph showing the results of fluorescence intensity growth rates corresponding to BRAF V600E (A) with different mutation abundances and EGFR L858R (B) with different mutation abundances based on Endo IV and DNAzyme provided by the embodiments of the present invention; Figure 8C- D is the linear fitting diagram of the increase rate of fluorescence intensity corresponding to different mutation abundances; Fig. 8A-D also inserts the corresponding fluorescence growth results of low mutation abundances.

Fig. 9 is the corresponding results of EGFR T790M (A), EGFR G719S (B) with different abundances and NRAS Q61R (C) with different abundances based on Endo IV and DNAzyme provided by the embodiment of the present invention. The result graph of the growth rate of fluorescence intensity; Figure 9D-F is the linear fitting graph of the increase rate of fluorescence intensity corresponding to different mutation abundances; Figure 9A-F also inserts the corresponding fluorescence growth results of low mutation abundance.

Figure 10 is a diagram of the abundance results of EGFR L858R determined by the Sanger method provided by the embodiment of the present invention; Figure 10A is the Sanger sequencing result of the EGFR L858R mutation obtained from the tissues of patients with non-small cell lung cancer; Sanger sequencing results of wild-type genomic DNA; Figure 10C is the Sanger sequencing results of synthetic L858R-103WT.

Figure 11 shows the Sanger sequencing results of 5 samples with different abundances (Sample 1=28%, Sample 2=15%, Sample 3=9%, Sample 4=1%, Sample 5=0.1%).

Figure 12 is a standard curve for the detection of EGFR-L858R (T>G) point mutations detected by the SNV two-step detection method based on Endo IV and DNAzyme.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Endo-nuclease IV (Endo IV) is a kind of abasic site (AP site) in double-stranded DNA (dsDNA), which can cut the phosphodiester bond upstream of the 5' direction of the AP site to generate a 3'OH terminal and 5' deoxyribose terminus (5'dRP), as shown in Figure 1A. In addition, Endo IV can also identify mismatched bases near the AP site, which has been used to detect SNVs. The basic principle is shown in Figure 1B. In short, it is to design an Endo IV probe (FP) containing an AP site, and label fluorescent and quenching groups at both ends of the probe. The sequence of FP is a perfect match with MT, but there is one base mismatch with WT. FP can form a first substrate (FP/MT) and a second substrate (FP/WT) with a complete double-stranded structure with MT and WT, respectively, while Endo IV has different cuts for the first substrate and the second substrate Quantitative detection of SNV can be realized by calculating the reaction rate difference between the two. However, this process needs to improve the sensitivity and selectivity of SNV detection by optimizing the reaction temperature and introducing auxiliary chains.

In the embodiment of the present invention, by exploring the cleavage activity of Endo IV for different dsDNA structures, it is found that Endo IV has the highest cleavage activity for perfectly matched dsDNA in the prior art. High cleavage activity, and through the verification of different DNA strands, it is found that this property is universal. That is, by designing the partial sequence of the probe and the mutant strand to form a truncated double-stranded sequence dsDNA, and this truncated sequence is located near the AP site, which can promote Endo IV to this truncated double-stranded sequence It has higher activity and thus cleaves the AP site more efficiently.

Through the discovery of this property, it is only necessary to design a class of Endo IV probes that can be partially complementary to the sequence near the mutation site of the mutant chain, and this probe contains an AP site to achieve Endo IV cleavage, greatly reducing the EndoIV requires complete sequence-dependence of substrates for probe formation matching the mutant strand.

For this reason, the embodiment of the present invention discloses a probe for DNA point mutation detection or for distinguishing a DNA mutant strand and a DNA wild strand, the probe is a single nucleotide strand, and the probe has a The AP site digested by enzyme IV, the AP site divides the probe into the first sequence (as shown in Figure 2B, the sequence near the 5' end of the FP-1 chain) and the second sequence, as shown in Figure 2B, near the 3' end of the FP-1 chain end sequence).

The probe can form the first substrate (FP-1/C1-(+1) as in Figure 4) and the second substrate (FP-1/C1-(+1) as in Figure 4) and the second substrate (FP-1/ C1-(+1/-XM)), the second substrate has a mismatched base site, and the mismatched base site corresponds to the mutation site of the mutant chain. The first substrate and the second substrate respectively have a first activity and a second activity of being cleaved by endonuclease IV to form a free single chain having the second sequence, and the first activity is greater than the second activity.

Thus, according to the mutation site of the mutant chain, design an Endo IV probe that matches the vicinity of the mutation site, and promote the proximity of the AP site and the mutation site, that is, it can promote the formation of the Endo IV probe and the mutation chain. The first substrate, and the activity of the first substrate combined with Endo IV is much greater than the activity of the second substrate formed by the Endo IV probe and the wild chain and Endo IV, that is, the first activity is much greater than the second activity . Therefore, through the way that the probe and the mutant chain form substrates, not only can the mutant chain and the wild chain be significantly distinguished, but also sensitively detect the mutant chain or the mutant chain with extremely low abundance in some biological materials or samples. mutant.

In the embodiment of the present invention, in order to characterize the difference in activity of the substrates formed by the probe, a fluorophore is connected upstream in the 5' direction of the AP site on the first sequence of the probe, and a fluorescent group is connected on the second sequence of the probe. A fluorescent quencher is attached downstream of the AP site in the 3' direction. In this way, when the probe is combined with the first substrate formed by MT or the second substrate formed by WT, the first substrate or the second substrate can be cut by Endo IV to form a free single strand with the second sequence, And the free single chain has a fluorescent quenching group, so that the cleavage process can generate fluorescence. Furthermore, the fluorescence intensity generated by the cleavage of the first substrate formed by MT and the fluorescence intensity generated by the cleavage of the second substrate formed by WT were recorded every 15s, and the fluorescence intensity-time curves of the two were drawn, and the slope of the curve was calculated. , which can be characterized as the rise rate of the fluorescence intensity of the first substrate formed by MT and the second substrate formed by WT, respectively, and the discrimination factor (DF) is the first substrate formed by MT and the second substrate formed by WT The ratio of the rising rate of the fluorescence intensity, the larger the DF value, the more favorable the probe is for distinguishing MT from WT, so as to obtain high selectivity and sensitivity.

The following will be described in combination with specific embodiments.

Reagents and Instruments

Endo IV was purchased from Thermo Fisher Scientific (USA). ThermoPol reaction buffer was purchased from New England Biolabs (USA). 2xTaq PCR Mix was purchased from Sangon Bioengineering Co., Ltd. (China). Gold nanoparticles (AuNPs, 20 nm) were purchased from BBI Solutions (UK). All experiments were diluted with DNase/RNase deionized water from Tiangen Biochemical Technology Co., Ltd. The fluorescence intensity of the samples was measured with a Roto-Gene Q2 plex HRM instrument (QIAGEN, Hilden, Germany). DNA-functionalized AuNPs were characterized using a transmission electron microscope (TEM) (JEM-1400Flash, JEOL, Japan), an ultraviolet-visible spectrometer (UV-3600, Shimadzu, Japan), and Zetasizer Nano S90 S90 (Malvern, UK). Agarose gel image analysis was performed on Bio-Rad ChemDocXRS (USA).

Probe, MT and WT synthesis

Table 1 and Table 2 list the nucleic acid sequences of the probes, MT and WT used in the embodiments of the present invention.

In Table 1, the fluorophore (FAM) and quencher (BHQ-1) labeled fluorescent probes used to explore the properties of Endo IV are named FP-1 and FP-2. The oligonucleotide chain complementary to FP-1 or FP-2 is designated as C1 or C2 sequence, respectively.

Table 1

Note: "□" indicates the AP site; the underlined base is the mismatch site with FP-1 or FP-2

In Table 2, the substrates of DNAzymes are named D-Sub-1 and D-Sub-2. D-Sub-1 can be labeled on AuNPs. D-Sub-2 is labeled with FAM and BHQ-1 to reflect the DNAzyme behavior in solution with fluorescent signals. DP-1 to DP-6 contain AP sites and DNAzyme sequences as the above-mentioned types of Endo IV probes. DP-1 was used to verify its feasibility as the above-mentioned type of Endo IV probe, and DP-2 to DP-6 were used to detect five different SNV probes.

Table 2

Note: "□" indicates the AP site; underlined is the general DNAzyme sequence; the base in bold is the mismatch site between the chain and DP-X

Based on Endo FP probe designed with IV substrate preference

Endo IV is an allopurine/uracytosine (AP) endonuclease. It acts on various oxidative damages in DNA and plays an important role in the base excision repair (BER) process in organisms. As shown in Figure 2A, Endo IV can cleave the AP site, and cut the DNA strand with the AP site (the red strand in the figure) into two parts. According to the prior art, the preferred substrate of Endo IV is a precisely matched dsDNA containing an AP site, and Endo IV exhibits the highest cleavage rate for this type of substrate. However, in the examples of the present invention, it was found that Endo IV exhibited different cleavage activities on dsDNA substrates with different structures.

In order to verify this different cleavage activity, the embodiment of the present invention carried out the following experiments:

1. Design FP chain

An AP-ssDNA probe containing 37 nucleotides (37nt) was designed, the sequence of which is FP-1 in Table 1. FP-1 is labeled with a fluorophore (FAM, the ball at the 5' end of the target strand in Figure 1B) and a fluorescent quencher (BHQ-1, the ball at the 3' end in Figure 1B). The AP site (AP site in Figure 1) can divide FP-1 into a first sequence and a second sequence, wherein the first sequence is a sequence near the 5' end, and the second sequence is a sequence near the 3' end.

2. The FP strand forms a complementary position with the target strand

Design a 23nt target strand (Target strand in the figure), which can be strictly complementary to a sequence of FP-1. As shown in Figure 2, the AP site of the FP-1 chain is defined as the "0" position, the 5' end of the Target chain is defined as the target line, and the bottom of the truncated double-stranded sequence formed by the Target chain and FP-1 The number of bases in the distance between the AP site and the FP-1 chain in the 3' direction is defined as "+", and the base number in the 5' direction is defined as "-". In this way, the corresponding Target chain can be designed as C1-( X) Sequence (as shown in Table 1), where "X" is the number of bases from the target chain to the AP site, and "X" is a positive integer.

As shown in Table 1, the FP-1 chain and its corresponding target sequence C1 sequence were thus synthesized, and different C1 sequences can be complementary to different positions of the FP-1 chain as the Target chain in Figure 2 (Shanghai Sangong), And according to the above method, use Endo IV to detect SNV, draw the fluorescence increase rate-time curve, and calculate DF, the results are shown in Figure 2.

In the initial state, because the fluorescence distance of FAM is relatively close, it is quenched by BHQ-1 through fluorescence resonance energy transfer (FRET). Endo IV generally exhibits low activity against ssDNA probes but high activity against dsDNA substrates. As shown in Figure 2B, once the AP site is cleaved by Endo IV, the BHQ-1-labeled fragment (7nt) will be cleaved to form a free fragment with the second sequence, thereby generating a fluorescent signal. Therefore, the increase rate of fluorescence intensity can reflect the activity of Endo IV on different substrates.

Figure 2C shows the different cleavage activities of Endo IV on different substrates formed by FP-1 and C1-(X) series. The results show that when the Target chain is C1-(-1), C1-(0), C1-(+1), C1-(2), C1-(3), C1-(4), C1-(5) , C1-(6) and C1-(7) each corresponded to a fluorescence rise rate more than 1 times the reaction rate corresponding to C1-T38, indicating that the FP-1 probe provided by the present invention does not need to utilize its full-length sequence and target strand Complementation, that is, having the activity of being cleaved by Endo IV, can be used as the basis for the design of EndoIV probes. This shows that the FP-1 provided by the embodiment of the present invention has the effect of more efficiently distinguishing the mutant chain and the wild chain, and has higher applicability. It can be seen that Endo IV has higher activity and preference for this truncated dsDNA substrate formed by FP-1. Among them, when the Target chain is C1-(+1), the reaction activity is the highest, and its fluorescence rise rate is 20 times that of C1-T38 (the target chain that is fully complementary to FP-1).

As shown in Table 1, another probe FP-2 and its corresponding Target chain C2 sequence were subsequently designed, and the same different C2 sequences can be complementary to different positions of FP-2 as the Target chain as shown in Figure 2 , to further verify the universality of the above properties. Similar results are also shown in Figure 3. According to the crystal structure of Endo IV and AP-DNA complex (Mi, L.; Sun, Y.; Shi, L.; Li, T. ACS Applied Materials & Interfaces 2020, 12, 7879-7887.), EndoIV needs to be in the AP site The point bends the dsDNA approximately 90° so that the AP site can access the active site of the enzyme for cleavage. Arginine 37 of the Endo IV enzyme stabilizes curved dsDNA by interacting with the base pair at the +1 position.

Thus, the substrate FP-1/C1(+1) has its truncated dsDNA structure so that it has lower energy required by Endo IV to induce bending of dsDNA during cleavage by Endo IV enzyme. Thus, a higher reaction rate is obtained compared to a perfectly matched target.

FP probe-based mutation detection

Since the activity of Endo IV enzyme-cutting the above-mentioned Endo IV probes such as FP is related to the AP site and the position change between the AP site and the complementary strand, this example further studies the relationship between the single nucleotide mutation site in the target chain and the above-mentioned Whether the truncated dsDNA formed by this Endo IV probe is cleaved is related to the enzyme activity.

In the above examples, because Endo IV exhibited the highest cleavage activity on FP-1/C1-(+1). As shown in Table 1, the C1-(+1) with 23-nt can be regarded as the MT chain; changing the MT chain corresponds to a base in C1-(+1) near the AP site, and different The C1-(+1/XM) chains were used as different WT chains respectively, and the C1-(+1/XM) sequences are shown in Table 1.

These MT strands and WT strands were combined with FP-1 to form truncated dsDNA structures, and were digested with Endo IV. The specific process was as follows: In a 200 μL PCR tube, 2 μL, 1 μM fluorescent probe (FP-1 or FP- 2), 2 μL, 1 μM target strand (C1 or C2 series), 2 μL of 10× ThermoPol reaction buffer and 2 μL of Endo IV (0.0025 U/μL) were added to the tube. The above samples were then diluted to 20 μL by deionized water. Finally, the tube was placed into a Roto-Gene Q2plexHRM instrument with a gain level of 7. Fluorescence intensity was measured every 15 seconds at 37°C, and the slope of the linear part of the fluorescence increase rate-time curve was the fluorescence growth rate. The discrimination factor (DF) was calculated using the ratio of fluorescence growth rates corresponding to MT and WT. All experiments were tested three times. Draw the fluorescence intensity-time curve, and calculate the above-mentioned rate and DF value of the fluorescence intensity. The result is shown in Figure 4.

As shown in Figure 4A, XM indicates that the X position in the dsDNA does not match that of FP-1. Since there is one base difference between C1-(+1/XM) and C1-(+1), C1-(+1/XM) can be regarded as WT, and C1-(+1) can be regarded as MT.

As shown in Figure 4B, when FP-1/C1(+1) is connected with FP-1/C1(+1/-1M), FP-1/C1(+1/-2M), FP-1/C1( The DF values between +1/-3M)FP-1/C1(+1/-4M) are 103, 7.6, 7.0 and 7.0 respectively, which can effectively distinguish WT chain and MT chain, therefore, FP-1 can effectively Utilize its AP site and the position relationship of the mutation site in the dsDNA structure complementary to the mutant chain to effectively distinguish the WT chain and the MT chain, and improve its selectivity as an SNV detection method. This is because the WT chain has a mismatched base, and this mismatched base is located near the AP site of this truncated dsDNA structure, so that the FP-1/C1-(+1/XM) The substrate is not conducive to the bending of dsDNA by the Endo IV enzyme, resulting in a reduced activity of its cleavage.

As shown in the corresponding Figure 4C, substrates are formed between FP-1 and the wild and mutant strands of C1-T38 with full complementary sequences, and the discrimination factors calculated corresponding to the cleaved fluorescence growth rates are not more than 1. , much smaller than the wild and mutant strands formed by FP-1 and partially complementary C1(+1).

Moreover, when the mismatch site is at the AP site-1 position, the activity is the lowest, and it has the greatest resistance to being bent by the Endo IV enzyme, so that FP-1/C1(+1) and FP-1/C1(+1/ -1M) corresponds to the largest DF value, which can most efficiently distinguish between WT chains and MT chains. Therefore, when actually detecting WT chains or mutants, FP chains can be designed facing each other, and based on known mutation sites, design an AP site with 1 base in the 5' direction of the mutation site, such as the FP in Figure 4A -1 probe, and the end point of the complementary position between FP-1 and the target strand is 1 base downstream of the 3' end of the AP site, so that FP probes can be designed according to different mutant chain sequences and their mutation sites, and then The probe is used to distinguish and detect mutant strands and wild strands, which greatly simplifies the freedom and universality of Endo IV probe design. Of course, the positional relationship between the mutation site of the FP probe and the AP site can also be several other relationships as shown in Figure 4, such as designing an AP site with 2, 3 or 4 bases in the 5' direction of the mutation site the FP probe.

This shows that the FP-1 probe disclosed in the embodiment of the present invention only needs to be partially complementary to the target strand to achieve efficient discrimination between the mutant strand and the wild strand, and is formed according to the mutation site and its AP site and the complementarity with the mutant strand. The positional relationship of mutation sites in the dsDNA structure can further enhance its discrimination effect and achieve higher selectivity and sensitivity for SNV detection.

DNAzymes and AuNPs

DNAzyme deoxyribozyme can catalytically hydrolyze the deoxynucleic acid sequence (D-Sub) on the substrate step by step along the microscopic molecular track, which is a kind of DNA that can catalyze the decomposition of DNA with enzyme-like activity, and is considered to be a useful signal amplification tool.

In order to overcome the problems of poor sensitivity and high cost, the embodiment of the present invention introduces DNAzyme as a general fluorescent signal generation platform, uses AuNPs as a substrate, and modifies D-Sub on the surface of AuNPs particles to form Sub-AuNPs as the catalytic molecular orbital of DNAzyme. And the D-Sub is marked with internal RNA base A (rA) and 3'-FAM.

When D-Sub binds to the surface of AuNPs via disulfide bonds to form Sub-AuNPs, AuNPs quench the labeled FAM on D-Sub. When the auxiliary ion Mn ²⁺ is added, Mn ²⁺ acts as a catalyst to catalyze DNAzyme to cut rA in Sub-AuNPs, and promote the nucleic acid fragments containing FAM to detach from the surface of AuNPs and emit fluorescent signals. Then, the DNAzyme breaks away from the cleaved fragment to bind another substrate, and the above cleavage process is repeated. Thus, one DNAzyme will cleave rA in multiple Sub-AuNPs and have the function of signal amplification.

The preparation method of D-Sub modified AuNPs is as follows:

According to the method published by Peng, H.; Li, X.-F.; Zhang, H.; Le, X.C. Nature Communications 2017, 8, 14378., the modified D-Sub-1 (see sequence in Table 2) was prepared AuNPs. Briefly, 5 μM D-Sub-1 was mixed with AuNPs for 2 hours at room temperature, then NaCl and Tween 20 were added to reduce non-specific adsorption and aggregation of AuNPs; after 24 hours of incubation, the above solution was centrifuged at 16000g After 20 min, D-Sub-1 modified AuNPs (Sub-AuNPs) were obtained after washing three times with 25 mM Tris-HCl (pH 7.4).

Characterization and quantification of Sub-AuNPs:

The product Sub-AuNPs were resuspended in Tris-HCl buffer solution and stored at 4 °C for further use. The D-Sub-1 modified AuNPs were correspondingly characterized using transmission electron microscopy (TEM), UV-vis spectroscopy, dynamic light scattering (DLS), zeta potential analysis, and agarose gel electrophoresis. Add 20 mM thiol to the prepared Sub-AuNPs solution and incubate overnight at room temperature. The fluorescence intensity of the supernatant was then measured after centrifugation. Subsequently, the standard curve of D-Sub-1 was used to calculate the concentration of D-Sub-1 loaded on AuNPs, and the results showed that, on average, each AuNP particle was covered by 295 pieces of D-Sub-1.

Sub-AuNPs were prepared by using the above examples, and their morphology and dispersion were observed by TEM. As shown in Figure 5A, both Sub-AuNPs and AuNPs particles were spherical with a diameter of 20nm. In Figure 5B, compared with unmodified AuNPs, the absorption peak of AuNPs was red-shifted from 525 nm to 530 nm due to the replacement of surface ligands of AuNPs by DNA. DNA on Sub-AuNPs also increased the hydrated particle size of AuNPs from 28.7 ± 3.4 nm to 48.7 ± 1.5 nm (Fig. 5C), and increased the zeta potential from -14.3 ± 0.2 mV to -7.8 ± 1.6 mV (Fig. 5D ). In addition, a 2% agarose gel electrophoresis experiment was performed at 70 V for 2 hours, and a decrease in the migration rate of Sub-AuNPs was observed (Fig. 5E). All these results confirm that the substrate of DNAzyme has been successfully attached to AuNPs.

Formation of DP probes based on DNAzyme and AuNPs

Although the FP probe designed based on Endo IV enzyme as provided in the above examples has high selectivity and sensitivity. However, when it is necessary to detect different mutant chains, it is still necessary to redesign probes labeled with fluorophores and fluorescent quenchers. The generally known fluorophores and fluorescent quenchers are expensive, and this will greatly The design cost of the probe is increased, thereby increasing the cost of the entire detection. Therefore, the embodiment of the present invention further optimizes the FP probe on the basis of the above, and replaces the downstream sequence of its AP site, that is, its second sequence with a DNAzyme sequence, and designs it as a DP probe to further improve its detection sensitivity, and discards the Fluorescent and quencher labels on the probes reduce detection costs.

Referring to the design of the FP chain in the above example, the second sequence in the FP chain is correspondingly replaced with a DNAzyme sequence to obtain a DP probe, thereby correspondingly synthesizing the probes of DP-1 and DP-2 (Shanghai Sangong) , the sequence is shown in Table 2.

As shown in Figure 6A, in this embodiment, the DNAzyme sequence (D-Sub-1, the part framed by the dotted line in the figure) is integrated on the first sequence of FP-1 (the part outside the dotted line frame in the figure), and the probe is removed. FAM and BHQ-1 labels on the needle, forming the DP-1 probe. The target strand (C1-(+1) (MT strand in the figure) as MT strand can hybridize with DP-1 to form truncated dsDNA, such as DP-1/C1-(+1).

This example found that due to the DNAzyme trapped in this truncated dsDNA structure, it is difficult to cut Sub-AuNPs with the help of cofactor Mn ²⁺ . The free DNAzyme can rapidly cleave rA on Sub-AuNPs under the catalysis of Mn ²⁺ , releasing FAM to generate an amplified fluorescent signal.

Therefore, by calculating the rise rate of fluorescence signals corresponding to different abundances of MT, a standard curve can be made, and the abundance of the mutant chain in the unknown sample can be obtained according to the standard curve.

Therefore, by designing the second sequence of the probe as a DNAzyme, the probe does not need to be modified with expensive fluorescent groups and quenching groups, so that the cost of probe optimization is significantly reduced, and the design rules are simple. At the same time, when detecting different target genes, only the red part of the probe chain needs to be replaced, the DNAzyme sequence remains unchanged, and the signal generation can be realized without replacing the fluorescent substrate chain on the gold nanoparticles.

DP probe-based SNV detection

Therefore, the embodiment of the present invention explores the feasibility of the SNV detection strategy based on Endo IV and DNAzyme. The target strand (MT(C1-(+1)) or WT(C1-(+1/-1M)) can hybridize with DP-1 to form a truncated dsDNA structure. The preferred substrate for Endo IV is DP-1 /MT, the AP site will be cleaved by Endo IV to release the free DNAzyme. Subsequently, the DNAzyme continuously cleaves the substrate on the Sub-AuNPs, resulting in an amplified fluorescent signal.

On the contrary, due to the weak cleavage activity of Endo IV and DP-1/WT, most of DP-1/WT is still not cleaved, so it can only obtain a very small amount of DNAzyme, which cannot react with the substrate on Sub-AuNPs to produce fluorescent signal. The principle is shown in Figure 7.

In the embodiment of the present invention, agarose gel electrophoresis was performed to verify the hybridization between DP-1 and the target chain (MT or WT), and the cutting behavior of Endo IV with DP-1/MT and DP-1/WT. As shown in Figure 7A, free DNAzyme and DP-1 showed a single band (lane 1 and lane 2). After DP-1/MT (3 lanes) or DP-1/WT (4 lanes) hybridization, the single-stranded band is brighter and slightly lagging compared to DP-1. When Endo IV was added to the DP-1/MT mixture, two new bands appeared, free DNAzyme (lane 5) and first sequence (lane 6). Compared with DP-1/MT, no other bands were observed in the DP-1/WT/Endo IV mixture, indicating that DP-1/WT has very low activity of being cleaved by Endo IV.

The detection based on Endo IV, DNAzyme and AuNPs can be divided into one-step detection (Figure 7B) and two-step detection (Figure 7C).

One-step detection:

1) In a 200 μL PCR tube, mix 2 μL of DP-1 (1 μM), 2 μL of the target strand (MT or WT, 2 μM) and 1.5 μL of ammonium sulfate (400 mM).

2) Continue to add 2 μL Endo IV, 2 μL Sub-AuNPs and 2 μL Mn ²⁺ (25 mM), and add deionized water to make the total volume reach 20 μL.

3) Put the PCR tube into the Roto-Gene Q2 plex HRM instrument and react at 37°C for 40min with a gain level of 9. Fluorescence intensity was measured every 15 s, and all experiments were performed independently and tested 3 times.

Figure 7B shows a one-step method for measuring SNV. Endo IV and Sub-AuNPs were added to the reaction system at the same time, and the results are shown in Figure 7D. Curve) and the fluorescence intensity of the WT chain (curve D) did not increase rapidly enough in the initial time.

Two-step detection:

1) In a 200 μL PCR tube, mix 2 μL DP-1 (1 μM), 2 μL target strand (MT or WT, 2 μM), 1.5 μL ammonium sulfate (400 mM) and 2 μL Endo IV.

2) After incubating at 37°C for 15 minutes, the above solution was heated at 80°C for 20 minutes to inactivate Endo IV.

3) Then add 2 μL of Sub-AuNPs and 2 μL of Mn ²⁺ (25 mM), deionized water to bring the total volume to 20 μL.

4) Put the PCR tube into the Roto-Gene Q2plex HRM instrument to react at 37°C for 40min, with a gain level of 9, and measure the fluorescence intensity every 15 seconds. All experiments were performed independently and tested 3 times.

When the two-step method shown in Figure 7C detects SNV, the first step is that the substrate formed by DP-1/C1-(+1) or DP-1/C1-(+1/-1M)) is cut by Endo IV, After the cleavage is completed, the second step is the catalytic reaction of DNAzyme on the substrate modified by AuNPs. The results are shown in Figure 7E. The increase in the fluorescence rate of MT and WT is significantly different from that in Figure 7E, especially in the initial stage (curve C ), thus having a more accurate detection effect.

Detection of 5 clinically relevant SNVs

The following example detected five synthetic clinically relevant SNVs (BRAF V600E, EGFR L858R, EGFR T790M, EGFR G719S and NRAS Q61R) based on DP probes, and the mutant and wild chains corresponding to the five SNVs are shown in Table 2 shown. These mutations are strongly associated with many cancers, such as NSCLC, colorectal and thyroid cancers. According to the mutation sites of these samples, five DNAzyme sequence-based probes DP-2-V600E, DP-3-L858R, DP-4-T790M, DP-5-G719S and DP-6-Q61R were designed in this example , the sequence is shown in Table 2.

As shown in Figure 8 and Figure 9, the detection limits of BRAF V600E, EGFR L858R, EGFR T790M, EGFR G719S and NRAS Q61R can reach 0.01%, 0.05%, 0.05%, 0.01% and 0.01%, respectively.

Table 3 The embodiment of the present invention provides the SNV detection comparison of the probe and other probes

As shown in Table 3, the lower limit of failure of general SNV detection methods based on Endo IV is between 0.003% and 1%. Among them, general Endo IV probes require temperature optimization or λ exonuclease assistance, and general Endo IV probes are more costly to design for different detection targets. In addition, the detection cost of the FP/DP probe disclosed in the present invention is 0.231$/tube, which is about one-tenth of the Sanger sequencing price of a sample and 1% of the price of a sample NGS sequence. Therefore, compared with other DNA probe methods, the detection limit of the mutation abundance of these SNVs based on the FP/DP probe method of the present invention is at the top level, and has the advantages of universality and simplicity.

EGFR-L858R(T>G) point mutation detection

In order to further demonstrate the potential clinical application of the probes provided in the examples of the present invention, in this example, tissue DNA was extracted from a patient with NSCLC for verification of clinical samples. The specific method is as follows:

1) The DNeasy Blood&Tissue Kit purchased by Tiantian Biotechnology Company was used to extract mutant EGFR L858R genomic DNA from non-small cell lung cancer (NSCLC) patient tissues, and the negative control group was commercial Promega genomic DNA.

2) Then use conventional PCR amplification to obtain 103-bp dsDNA containing EGFR L858R mutant and wild-type DNA, using forward primer L858R-FP (as shown in SEQ ID NO.67 in Table 2) and reverse primer L858R-RP (shown as SEQ ID NO.68 in Table 2), the extracted genome sample was amplified by PCR. And the abundance of EGFRL858R was estimated to be 53% by Sanger sequencing (Fig. 10).

3) According to the abundance level, the MT sample was diluted with a specified amount of normal genomic DNA from Promega (USA) to prepare standard samples with different mutation abundances (50%, 20%, 10%, 50%, 1%, 0.5 %, 0.05%, 0).

4) An asymmetric PCR reaction is then carried out to obtain a suitable ssDNA target strand. Finally, the abundance of EGFR L858R in the ssDNA target strand was measured by the method provided in the above examples. Specifically: in a 200 μL PCR tube, add 25 μL 2×Taq PCR Mix, 10 μL×5 μM L858R-FP, 1 μL×5 μM L858R-RP and more than 20 ng of standard samples, deionized water to make the total volume reach 50 μL, and in the Rotor-Gene Q2plex The PCR program (15s, 94°C, 50°C, 72°C, 15s, 35 cycles) was performed on the HRM instrument.

5) After the asymmetric PCR is completed, the TIANquick Mini Purification Kit is used to purify the target chain, and the concentration is determined using a NanoDrop 2000 UV-Vis spectrophotometer. Then the standard curve of the actual sample obtained according to (3). The actual sample concentration of unknown abundance can be deduced from the standard curve.

6) Then perform SNV mutation detection on (3) by a two-step method to obtain a standard curve, as shown in FIG. 12 . The sequences of L858R-103MT, L858R-103WT and DP-3-L858R probes involved are shown in Table 2.

In order to compare the two-step method provided by the present invention with the most widely used mutation detection method Sanger, another five standard samples (28%, 15%, 9%, 1% and 0.1%) with different mutation abundances were also prepared for this purpose , the results in Figure 11, Figure 12 and Table 4.

Table 4

Standard sample concentration Sanger method two step method 28% 29% 29.6% 15% 16% 16.3% 9% 10% 11.0% 1% not detected 1.2% 0.1% not detected 0.09%

As shown in Figure 12, the two-step method provided by the present invention detects as low as 0.05%. Table 4 shows the results of 5 different abundances of EGFR L858R mutation samples measured by the Sanger method and the two-step method provided in the examples of the present invention. As shown in Table 4, when the mutation abundance is greater than 10%, the abundance measured by Sanger and the method of the present invention is basically consistent with the initial abundance of the standard sample. Sanger cannot detect low-abundance mutation (<10%) samples, and the method disclosed by the invention can still detect low-abundance samples, and the detected abundance is consistent with the initial abundance in the standard sample, which shows that the disclosed method of the present invention The probe and the detection method using the probe have practicality in the detection of clinical samples.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention.

sequence listing

<110> Huazhong University of Science and Technology

<120> Probes, kits and applications for detecting DNA point mutations

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Claims (3)

1. a kind of probe, is used to distinguish the dna mutant chain and dna wild chain that contain single nucleotide mutation, it is characterized in that, described probe comprises: a single strand of nucleotides having an AP site cleaved by endonuclease IV that divides the probe into a first sequence and a second sequence; A fluorophore linked upstream in the 5' direction of the AP site on the first sequence; and A fluorescence quenching group connected downstream of the AP site 3' direction on the second sequence; The probe forms a first substrate with a first double-stranded sequence through its partial sequence and the mutant chain, and the mutation site of the mutant chain is in the first double-stranded sequence; The probe forms a second substrate with a second double-stranded sequence through its partial sequence and the wild strand, and the second substrate has a mismatched base site, and the mismatched base site The point corresponds to the mutation site; the first double-stranded sequence and the second double-stranded sequence correspond to the same sequence position; The 5' end of the mutant strand used to form the first double-stranded sequence is located 1 base downstream from the AP site in the 3' direction of the probe, and is used to form the second double-stranded sequence. The mismatched base site of the wild strand of the strand sequence is 1 base upstream from the AP site in the 5' direction of the probe; The first substrate has the first activity of being cleaved by endonuclease IV to form a free single chain having the second sequence, and the second substrate has the ability of being cleaved by endonuclease IV to form a free single chain having the second sequence. a second activity of the free single strand of the second sequence, the first activity being greater than the second activity; The first activity is characterized by the fluorescence rise rate generated during the process of the first substrate being cleaved by endonuclease IV to form a second sequence carrying a fluorescent quenching group; the second activity is characterized by the first Characterized by the fluorescence rise rate generated during the process of the second substrate being cleaved by endonuclease IV to form a second sequence carrying a fluorescent quenching group; The probe is shown in any one of SEQ ID NO 49, 50, 53, 56, 59, 62. 2. The test kit or reagent combination that is used for DNA point mutation detection is characterized in that, comprises the probe described in claim 1, endonuclease IV, nanoparticle and auxiliary Mn ²⁺ , and described nanoparticle is connected with label Fluorophores and substrates for DNAzyme cleavage; The probe comprises a single stranded nucleotide having an AP site cleaved by endonuclease IV, which divides the probe into a first sequence and a second sequence, the second The sequence is a DNAzyme sequence; The probe forms a first substrate with a first double-stranded sequence through the first sequence and the mutant chain, and the mutation site of the mutant chain is in the first double-stranded sequence; The probe forms a second substrate with a second double-stranded sequence through the first sequence and the wild strand, the second substrate has a mismatched base site, and the mismatched base The base site corresponds to the mutation site; the first double-stranded sequence and the second double-stranded sequence correspond to the same sequence position; The 5' end of the mutant strand used to form the first double-stranded sequence is located 1 base downstream from the AP site in the 3' direction of the probe, and is used to form the second double-stranded sequence. The mismatched base site of the wild strand of the strand sequence is 1 base upstream from the AP site in the 5' direction of the probe; The first substrate has the first activity of being cleaved by endonuclease IV to form a free single chain having the second sequence, and the second substrate has the ability of being cleaved by endonuclease IV to form a free single chain having the second sequence. a second activity of the free single strand of the second sequence, the first activity being greater than the second activity; The first activity is characterized by the fluorescence rise rate generated during the process of cutting and connecting the substrate on the gold nanoparticle by the free DNAzyme formed by the first substrate being cleaved by endonuclease IV; the second The activity is characterized by the rate of increase in fluorescence generated during the process of cleavage of the free DNAzyme formed by the cleavage of the first substrate by endonuclease IV and connection of the substrate on the gold nanoparticles. 3. The test kit or reagent combination described in claim 2 detects the application in the sample that contains the mutated chain of DNA point mutation and wild chain, and this application does not comprise the treatment and the diagnosis of disease, and described mutated chain is selected from BRAF V600E, One of EGFR L858R, EGFR T790M, EGFR G719S or NRAS Q61R.