A method for detecting double-stranded DNA mismatch type and position

Abstract

The present invention belongs to the field of gene detection technology, and discloses a method for detecting the type and position of double-stranded DNA mismatch, which comprises the following steps: after combining a double-stranded DNA sample and a ligand that can selectively bind to a specific type of base mismatch site, the thermal stability difference index before and after the double-stranded DNA sample is combined with the ligand is detected by an α-hemolysin nanopore detection platform to obtain the type and position information of the mismatched base of the double-stranded DNA. At the single-molecule level, the present invention directly uses the thermal stability difference of the double-stranded unwinding time before and after the addition of the ligand to obtain information about the mismatch and the type and position of the mismatch of the double-stranded DNA, which has a significant time advantage compared with the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a method for detecting the type and position of double-stranded DNA mismatch. Background Art

[0002] Gene mutations pose a huge threat to global health and sustainable development. Rapid diagnosis of the type of gene mutation and the location of mismatches has always been the goal pursued in the field of biosensors. Although there have been many reports on excellent DNA detection biosensors, including sensors based on sensing principles such as PCR amplification, microarrays, fluorescence, and electrochemistry, single-molecule resolution nanopore technology still shows non-competitive advantages in accuracy and specificity, especially for protein nanopores, which can also be developed into in vitro diagnostic (IVD) platforms in addition to time-consuming DNA sequencing purposes. In recent years, in-depth research has been conducted on targets such as small molecules, miroRNA, DNA, peptides, and proteins, revealing the powerful characteristics of nanopore technology.

[0003] Although many existing literatures have reported the detection of single-base mismatches in DNA duplexes and the detection of single-base mutations, base modifications and excisions of microRNAs, it is still difficult to find a simple method to simultaneously obtain information about mismatches and mismatch types without time-consuming DNA sequencing using protein nanopores. The latest research reports indicate that since the diameter of the locking region in the vestibule of the α-HL nanopore is slightly larger than the cross-section of the double-stranded DNA, single mismatches including CA, CT, TC, etc. can be distinguished by the interaction between the mismatched base pairs and the locking region; however, the lower concentration salt environment used is required for a larger current signal, which sacrifices the sensitivity of nanopore detection. In addition, this method requires that the position of the double-stranded mismatch pair must be close to the locking region, which hinders flexible design and wide application. Therefore, it is very necessary to design a simple method that is not subject to too many restrictions and can detect the type and position of double-stranded DNA mismatches. Summary of the invention

[0004] In response to the above problems, the present invention provides a method for detecting the type and position of double-stranded DNA mismatches. At the single-molecule level, the difference in thermal stability of the double-stranded unwinding time before and after the addition of a ligand is directly used to obtain information about the mismatches of the double-stranded DNA and the type and position of the mismatches, so as to solve the above problems.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A method for detecting the type and position of double-stranded DNA mismatches, specifically combining a double-stranded DNA sample and a ligand that can selectively bind to a specific type of base mismatch site, and then detecting the thermal stability difference index before and after the double-stranded DNA sample binds to the ligand through an α-hemolysin nanopore detection platform to obtain the type and position information of the mismatched bases in the double-stranded DNA.

[0007] α-Hemolysin (αHL) is a water-soluble protein monomer with a molecular weight of 33.2kD secreted by Staphylococcus aureus. It can be assembled into a mushroom-shaped heptamer protein nanopore with a molecular weight of 232.4kD on the phospholipid bilayer. The total length of the pore is about 10nm. The nanopore structure is stable and can be completely loaded in the bilayer lipid membrane without leakage. It is an ideal nanopore detection device. The α-hemolysin nanopore has two sensitive areas: the closed area of ​​the vestibule and the central contraction area of ​​the β-barrel. Using double-stranded DNA labeled with two single-stranded long tails as a prototype, the overall sensing mechanism of the α-HL nanopore can be explained. Driven by the transmembrane voltage, negatively charged DNA at the single-molecule level is first captured by the vestibule of the α-HL nanopore, and then the single-stranded tail enters the narrow channel of the β-barrel through the central contraction; since the latch region and the central contraction region are the narrowest parts of the vestibule and β-barrel in the α-HL nanopore, respectively, both of them are very sensitive to local environmental fluctuations in the solution. Therefore, the latch region is generally used to detect subtle changes in double-stranded DNA, including nick sites, base pair mismatches, nucleoside substitutions and enzyme activity; the central contraction region with a diameter of less than 2nm can prevent the translocation behavior of double-stranded DNA and force the double-stranded DNA to unwind, and its unwinding time is highly dependent on the thermal stability of the double-stranded DNA. Therefore, its unwinding time can be used to effectively distinguish other targets such as mismatched base pairs, small ligand binding, peptides, etc.

[0008] In some embodiments of the present invention, the specific type of base mismatch site includes GG, GT, GA, CC, AA, TT, CA, CT or TC. In some embodiments of the present invention, when the specific type of base mismatch site is GG, the ligand is 2-amino-1,8-naphthyridine dimer.

[0009] In recent years, researchers have synthesized ligands with specific binding ability to mismatches such as GG, GT, and AA, which can effectively change the thermal stability of double-stranded DNA and thus affect the unwinding time through the protein nanopore; according to literature reports, 2-amino-1,8-naphthyridine dimer can selectively bind to GG mismatches, and the dissociation constant after binding becomes 53nM, thereby significantly improving the thermal stability of double-stranded DNA containing GG mismatches.

[0010] In some embodiments of the present invention, the thermal stability difference indicator is the unwinding time of the double-stranded DNA when passing through the α-hemolysin nanopore.

[0011] In some embodiments of the invention, the unwinding time includes the dwell time and / or blocking current of each translocation event.

[0012] In some embodiments of the present invention, the method for preparing the α-hemolysin nanopore comprises the following steps:

[0013] Diphenylphosphatidylcholine dissolved in decane is added into the bilayer chamber, and an electrolyte solution is injected at the orifice to form a planar lipid bilayer membrane through DPhPC. α-HL protein is added into the cis cavity, and the cis cavity is connected to the "ground". A voltage of 200 mV is applied between the two chambers through two Ag / AgCl electrodes, and the protein is moved to be inserted into the phospholipid bilayer.

[0014] In some embodiments of the present invention, the method for detecting the thermal stability difference index comprises the following steps:

[0015] Double-stranded DNA or its mixture with ligand is added to the cis chamber, and the change of current is recorded by patch clamp amplifier to obtain the data of translocation signal peak. The duration of translocation signal is statistically analyzed to obtain the mismatch base and position information of double-stranded DNA.

[0016] The beneficial effects of the present invention are:

[0017] The method provided by the present invention can explore the difference in the unwinding characteristics of double-stranded DNA before and after binding to a ligand through a protein nanopore at a single-molecule level, and directly use the difference in thermal stability of the double-stranded unwinding time before and after the addition of the ligand to obtain information about the mismatch and mismatch type and position of the double-stranded DNA. Specifically, the decompression ability of the α-HL protein nanopore is used to detect the double-stranded DNA after the action of the ligand small molecule. If there is a specific type of base mismatch, it can be observed that the duration average value of the blocking current of the double-stranded DNA after the action of the small molecule is significantly shifted toward the value corresponding to the fully complementary DNA; if there is no specific base mismatch, there is no obvious change. Based on this, the present invention directly distinguishes the mismatch type and approximate position through the change law of the duration. Compared with the prior art, it has obvious time advantages. The entire detection process only takes a few minutes to determine, and the test solution and the test current do not need to be specifically adjusted, which is the same as the general nanopore test process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0019] Figure 1 Schematic diagram of the principle of the method for detecting the type and position of double-stranded DNA mismatch according to an embodiment of the present invention;

[0020] Figure 2 1 is a translocation signal diagram of double-stranded DNA containing GG mismatch and complete pairing before and after adding 2-amino-1,8-naphthyridine dimer ligand in Example 1 of the present invention, wherein A is a double-stranded DNA containing GG mismatch base; B is a double-stranded DNA containing GG mismatch base and adding ligand molecule; C is a double-stranded DNA with complete complementary pairing; D is a double-stranded DNA with complete complementary pairing and adding ligand molecule;

[0021] Figure 3 : is a histogram of changes in the double-stranded DNA pore dwelling events containing other mismatch types in the embodiment of the present invention before and after the addition of 2-amino-1,8-naphthyridine dimer; wherein A is before addition; B is after addition;

[0022] Figure 4 : is a translocation signal diagram of the DNA double strand containing GG mismatch (at one quarter) before and after the addition of 2-amino-1,8-naphthyridine dimer ligand in Example 2 of the present invention, wherein A is before the addition of ligand; B is after the addition of ligand;

[0023] Figure 5 1 is a graph of corresponding via signals of different mutation numbers before and after adding 2-amino-1,8-naphthyridine dimer ligand in the embodiment of the present invention, wherein A is a double site; B is a triple site;

[0024] Figure 6 3 is a comparison diagram of the event noise after the double-stranded binding of the ligand small molecule at the intermediate mutation and the quarter mutation site in the embodiment of the present invention, wherein A is the intermediate mutation; B is the quarter mutation. DETAILED DESCRIPTION

[0025] The present invention is further described in conjunction with the following examples.

[0026] Example 1

[0027] The method for detecting the mismatch type of double-stranded DNA provided in this embodiment comprises the following steps:

[0028] (1) Incubation of double-stranded DNA with ligand;

[0029] All DNAs (nucleic acid sequences are shown in Table 1 ) were dialyzed in 10 mM potassium phosphate buffer (pH 6.2) for more than 12 h, and double-stranded probes were prepared in hybridization buffer (10 mM Tris, 100 mM NaCl, pH 7.4). The solution was incubated at 90 °C for 5 min and then cooled to room temperature;

[0030] Table 1 Nucleotide sequences used in this example

[0031] Name Sequence <![CDATA[P C ]]> 5'-GTCGCGATCTTGGTG-3' <![CDATA[P 30-G ]]> 5'-C30-CACCAAGATCGCGAC-C30-3' <![CDATA[P G ]]> 5'-GTCGCGATGTTGGTG-3' <![CDATA[P 30-C ]]> 5'-C30-CACCAACATCGCGAC-C30-3' <![CDATA[P T ]]> 5'-GTCGCGATTTTGGTG-3' <![CDATA[P 30-T ]]> 5'-C30-CACCAATATCGCGAC-C30-3' <![CDATA[P A ]]> 5'-GTCGCGATTATGGTG-3' <![CDATA[P 30-A ]]> 5'-C30-CACCAAAATCGCGAC-C30-3' <![CDATA[P 30-2M ]]> 5'-C30-CACCAAGATCGGGAC-C30-3' <![CDATA[P 30-3M ]]> 5'-C30-CACGAAGATCGGGAC-C30-3'

[0032] (2) Fabrication and data collection of protein nanopores

[0033] Diphenylphosphatidylcholine (10 mg / mL) dissolved in decane was added to a 0.5 mL double-layer chamber, and an electrolyte solution was injected at the 100 μm orifice to form a planar lipid bilayer membrane through DPhPC. α-HL protein (5 μg / mL) was added to the cis chamber, and the cis chamber was connected to the "ground". A voltage of 200 mV was applied between the two chambers through two Ag / AgCl electrodes, and the protein was waited to move to insert into the phospholipid bilayer. After a single α-HL nanopore was inserted into the lipid bilayer, the probe, target DNA or a mixture thereof (the final concentration of each probe or target was 200 nM) was added to the cis chamber, and the change in current was recorded with a patch clamp amplifier to obtain the data of the translocation signal peak, and the duration of the translocation signal was statistically analyzed to obtain the mismatch base and position information of the double-stranded DNA.

[0034] All experiments were performed in 1.0 M KCl (10 mM Tris and 0.1 mM EDTA, pH 8.0) solution, with a voltage of +120 mV and a temperature of 22 ± 2°C.

[0035] As attached Figure 2 As shown in A and 2B, before the addition of the ligand small molecule 2-amino-1,8-naphthyridine dimer, the DNA double helix containing GG mismatch (the mismatch position is in the middle), the current time trajectory shows normal event characteristics, and the median residence time is 56ms, which is consistent with previous literature reports; after adding 2-amino-1,8-naphthyridine dimer, the median residence time is 352ms, and the duration of the event is significantly increased, indicating that after the addition of 2-amino-1,8-naphthyridine dimer, a strong bond is generated between the GG mismatches.

[0036] As attached Figure 2As shown in C and 2D, for DNA duplexes without GG mismatches inside, the median event duration of the samples before and after the addition of 2-amino-1,8-naphthyridine dimer hardly moved, both being around 420 ms, indicating that the presence of 2-amino-1,8-naphthyridine dimer has no effect on the permeation of the fully complementary paired DNA duplexes; in addition, the median residence time of the duplexes containing GG base mismatches after the addition of small molecules is similar to that of the fully complementary paired DNA duplexes (352 and 420 ms), thus verifying that the addition of 2-amino-1,8-naphthyridine dimer enhances the thermal stability of the duplexes containing GG mismatches and has similar thermal stability to the fully complementary paired double-stranded DNA.

[0037] The detection method described in this embodiment shows good selectivity for the type of base mismatch. Figure 3 As shown, after adding 2-amino-1,8-naphthyridine dimer, only the dwell events of the duplex containing GG base mismatch increased significantly (from 56ms to 352ms), while all other types of mismatches: GA mismatch (98ms vs. 128ms), GT mismatch (62ms vs. 66ms), CC mismatch (29ms vs. 38ms), AA mismatch (49ms vs. 62ms), and TT mismatch (32ms vs. 29ms) did not show any reaction.

[0038] Example 2

[0039] The method for qualitatively detecting the position of mismatched bases in double-stranded DNA provided in this embodiment is the same as that in Example 1. Figure 4 As shown, the duration of DNA double-stranded pore passage when the GG mismatched base pair is located at one-quarter of the double-strand is very short, with a median residence time of 17ms; after adding 2-amino-1,8-naphthyridine dimer, the median residence time reaches 864ms; compared with the data of Example 1 in which the mutation site is in the middle of the double-strand, it can be seen that before and after the addition of 2-amino-1,8-naphthyridine dimer, the change in the median residence time of the double-stranded DNA with a GG mismatch at one-quarter of the double-strand is much greater than the value corresponding to the DNA duplex with the GG mutation site located in the middle of the double-strand (57ms to 352ms), which may be due to the fact that the mutation site is closer to the locking region of the nanopore at one-quarter, resulting in a more sensitive perception of the binding force change; therefore, the experimental data show that regardless of whether the mutation site is in the middle or one-quarter of the double-strand, 2-amino-1,8-naphthyridine dimer can enhance the thermal stability of the DNA double-strand, but is more sensitive to the time change caused by the GG mismatched base pair closer to the locking region in the DNA double-strand.

[0040] As attached Figure 4As shown, in DNA duplexes with two GG mismatches (one quarter and one half) and three GG mismatches (one quarter and one half at both ends), after adding 2-amino-1,8-naphthyridine dimer, the median residence time of the multiple mismatched DNA duplexes can be well separated from the median residence time of the same DNA duplexes.

[0041] Finally, by carefully examining the event features in the current traces, the events generated by the ligand-bound mismatched double-stranded DNA during unwinding through the nanopore showed higher current fluctuations and shorter spikes than those without ligand. Figure 6 The enlarged view of the graph shows that the unwinding noise level after ligand binding for the GG mispaired duplex near the latch region (one quarter of the duplex) is significantly higher than that for the GG mismatched duplex in the middle of the duplex. All these observations can be attributed to the interaction of the duplex with the sensitive latch region. In addition, due to the high concentration of the small ligand in the buffer solution, all the short peaks can be assigned to the transport of small molecules through the protein nanopore. In general, at this stage, only qualitative identification in terms of noise level can be provided.

[0042] The above embodiments of the present invention directly utilize the difference in thermal stability of double-stranded DNA unwinding time before and after the addition of ligands to obtain information about mismatches and mismatch types and locations of double-stranded DNA at the single-molecule level, which has obvious time advantages compared to the prior art.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

[0044]

[0045]

[0046]

Claims

1. A method for detecting the type and position of double-stranded DNA mismatch, characterized in that: After combining a double-stranded DNA sample and a ligand that can selectively bind to a specific type of base mismatch site, the thermal stability difference index before and after the double-stranded DNA sample binds to the ligand is detected by an α-hemolysin nanopore detection platform to obtain the type and position information of the mismatched base of the double-stranded DNA; The specific type of base mismatch site is GG, and the ligand is 2-amino-1,8-naphthyridine dimer; The thermal stability difference index is the unwinding time of the double-stranded DNA when passing through the α-hemolysin nanopore; The unwinding time includes the dwell time and / or blocking current of each translocation event.

2. A method for detecting double-stranded DNA mismatch types and positions according to claim 1, characterized in that: The preparation method of the α-hemolysin nanopore comprises the following steps: Diphenylphosphatidylcholine dissolved in decane was added into the bilayer chamber, and the electrolyte solution was injected at the orifice to form a planar lipid bilayer membrane through DPhPC. The α-HL protein was added into the cis cavity, and the cis cavity was connected to the "ground". A voltage of 200 mV was applied between the two chambers through two Ag / AgCl electrodes, and the protein was moved to be inserted into the phospholipid bilayer.

3. A method for detecting double-stranded DNA mismatch types and positions according to claim 1, characterized in that: The method for detecting the thermal stability difference index comprises the following steps: Double-stranded DNA or its mixture with ligand is added to the cis chamber, and the change of current is recorded by patch clamp amplifier to obtain the data of translocation signal peak. The duration of translocation signal is statistically analyzed to obtain the mismatch base and position information of double-stranded DNA.

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