WO2018196691A1 - Procédé de reconnaissance précise pour acide nucléique - Google Patents

Procédé de reconnaissance précise pour acide nucléique Download PDF

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WO2018196691A1
WO2018196691A1 PCT/CN2018/083921 CN2018083921W WO2018196691A1 WO 2018196691 A1 WO2018196691 A1 WO 2018196691A1 CN 2018083921 W CN2018083921 W CN 2018083921W WO 2018196691 A1 WO2018196691 A1 WO 2018196691A1
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nucleic acid
hybridization probe
target nucleic
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hybridization
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倪燕翔
牛钢
马紫珊
曹博
张奇伟
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倪燕翔
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Definitions

  • the present disclosure relates to the field of biometrics, and in particular to a method for accurately identifying nucleic acids.
  • FISH fluorescence in situ hybridization
  • Non-Patent Document 2 proposes to label a chromosomal region containing a repeat sequence with a DNA probe containing a 2.1 kb HaeIII fragment, and obtain an image having a resolution of about 50 nm by photo-activated localization microscopy (PALM). .
  • PAM photo-activated localization microscopy
  • Patent Document 1 discloses a super-resolution imaging method.
  • the docking strand is hybridized to the target molecule to be recognized, and then the fluorescently labeled imager strand is bound to the docking strand and then imaged by activating the fluorophore on the imager strand.
  • This method requires the use of at least two functional molecules (docked strands and imager strands), which is cumbersome to operate and detrimental to the efficiency of labeling of the target molecule.
  • DNA paint is used as a docking chain.
  • the DNA paint is a marker that marks the entire length of the chromosome region to be recognized (Patent Document 2). Users need to build complex systems to prepare DNA coatings, and in the process of preparing DNA coatings by PCR, the final concentration of the prepared DNA coatings may be deviated due to sequence preference, resulting in instability of the FISH label.
  • DNA coatings can produce non-specific binding to the sample to be tested, resulting in a decrease in image quality.
  • it is necessary to increase the temperature at which the DNA coating hybridizes with the sample.
  • an increase in temperature in turn leads to a decrease in the intensity of the specific hybridization required, and thus a greater number of imaging strands are required to effectively label the target molecule to be tested. This hinders further improvement in resolution, and thus it is difficult to achieve high-resolution imaging of shorter target molecular regions.
  • Patent Document 1 WO 2015/017586
  • Patent Document 2 US 2010304994 (A1)
  • Non-Patent Document 1 Langer-Safer PR, Levine M, Ward DC. Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci U S A. 1982 Jul; 79(14): 4381-5.
  • Non-Patent Document 2 Weiland, Y., Lemmer, P., Cremer, C. 2011. Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures. Chromosome Res, 19, 5-23.
  • Non-Patent Document 3 Rouillard, J.M., Zuker, M. & Gulari, E. 2003.
  • OligoArray 2.0 design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res, 31, 3057-62.
  • Non-Patent Document 4 Markham, N.R. & Zuker, M. 2008. UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol, 453, 3-31.
  • Non-Patent Document 5 Huang, B., Wang, W., Bates, M. & Zhuang, X. 2008. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science, 319, 810-3.
  • DNA means deoxyribonucleic acid.
  • RNA means ribonucleic acid
  • Molecular beacon means an oligonucleotide fragment of a stem-loop structure in which a fluorophore and a quencher are respectively conjugated to the 5' and 3' ends, the molecular beacon being in the following two states The morphology is different to produce an identifiable signal: (i) the molecular beacon is freely bound to the target nucleic acid, and (ii) the molecular beacon is bound to the target nucleic acid.
  • T m value indicates the temperature at which the double-stranded structure of the double-stranded nucleic acid is dissociated by half.
  • the object of the present disclosure is to provide a method for identifying a nucleic acid, which not only simplifies the step of nucleic acid recognition, but also efficiently marks a target nucleic acid molecule, thereby improving the efficiency and accuracy of recognition.
  • the present disclosure provides a method for identifying a nucleic acid, the method comprising the steps of:
  • the hybridization probe comprises a reporter group and the hybridization probe is in a different state in two cases to generate an identifiable signal by the reporter group: (i) the hybridization probe is at In the free state, (ii) the hybridization probe binds to the target nucleic acid.
  • step c of the identification method is performed by fluorescence spectrophotometer reading or fluorescence microscopy imaging, preferably the fluorescence microscopy imaging is super-resolution imaging.
  • a hybridization probe comprises a binding region and a dissociation region, the dissociation region self-hybridizing when the hybridization probe is in a free state, the reporter group not emitting an identifiable signal;
  • the hybridization probe specifically binds to a target sequence fragment of the target nucleic acid by the binding region, the dissociation region dissociates and the reporter group emits an identifiable signal.
  • the reporter group comprises a fluorophore and a quencher, the identifiable signal being fluorescent; preferably the fluorophore is Alexa-647, and/or the quencher is BHQ3.
  • the hybridization probe comprises a loop structure serving as the binding region, and at least two arm structures serving as the dissociation region, wherein the at least two arm structures are respectively located in the loop structure On both sides.
  • the loop structure has a length of 20 nt to 60 nt, preferably 25 nt to 55 nt, more preferably 30 nt to 50 nt, still more preferably 35 nt to 45 nt, still more preferably 40 nt to 44 nt, and/or the arm structure
  • the length is 4 nt to 10 nt, preferably 5 nt to 9 nt, preferably 6 nt to 8 nt, more preferably 7 nt.
  • sequence identity between the loop structure and the target sequence fragment of the target nucleic acid is from 90% to 100%, preferably from 95% to 100%, more preferably from 98% to 100%.
  • the target nucleic acid has a length of less than 4.9 kb; preferably, the target nucleic acid has a length of 3.3 kb or less, more preferably 2.5 kb or less.
  • the target nucleic acid does not contain a repeat sequence.
  • the step a of the identification method is carried out by incubating the sample with the hybridization probe at a temperature of 70 ° C to 80 ° C, preferably 73 ° C to 77 ° C, more preferably 75 ° C, Then, the hybridization is carried out at a temperature of 10 ° C to 42 ° C, more preferably 12 ° C to 38 ° C, more preferably 14 ° C to 34 ° C, still more preferably 16 ° C to 30 ° C, still more preferably 18 ° C to 26 ° C, still more preferably 22 ° C.
  • the standard deviation of the single-molecule repeat positioning accuracy of the super-resolution imaging in the xy direction is 50 nm or less, preferably 20 nm or less, further preferably 9 nm or less; or the super-resolution imaging is in the xy direction
  • the full width at half maximum of the single molecule repeat positioning accuracy is 120 nm or less, preferably 50 nm or less, and more preferably 20 nm or less.
  • the standard deviation of the single-molecule repeat positioning accuracy of the super-resolution imaging in the z direction is 100 nm or less, preferably 40 nm or less, further preferably 20 nm or less; or, the super-resolution imaging single molecule in the z direction
  • the full width at half maximum of the repeat positioning accuracy is 250 nm or less, preferably 100 nm or less, and more preferably 50 nm or less.
  • the laser power used when exciting the fluorophore is 0.80 kW/cm 2 to 2.00 kW/cm 2 , preferably 0.90 kW/cm 2 to 1.45 kW/cm 2 , further preferably 1.00 kW/cm 2 . .
  • the sampling frame rate when the fluorescence is recognized is 10 Hz or more, preferably 50 Hz or more, more preferably 60 Hz or more, further preferably 85 Hz or more.
  • the present disclosure also provides the use of a hybridization probe for fluorescence spectrophotometric reading or fluorescence microscopy imaging of a target nucleic acid, wherein the hybridization probe comprises a reporter group, and the hybridization probe is in two cases The state is different such that an identifiable signal is produced by the reporter group: (i) the hybridization probe is in a free state, and (ii) the hybridization probe binds to the target nucleic acid.
  • the fluorescent microscopic imaging is super-resolution imaging; preferably the target nucleic acid has a length of less than 4.9 kb, for example a length of 3.3 kb or less, such as 2.5 kb or less; preferably the target nucleic acid does not contain a repeat sequence;
  • the standard deviation of the single-molecule repeat positioning accuracy of the super-resolution imaging in the xy direction is 50 nm or less, for example, 20 nm or less, for example, 9 nm or less; or, the full-width and full width of the single-molecule repeat positioning accuracy of the super-resolution imaging in the xy direction is 120 nm.
  • the standard deviation of the single-molecule repeat positioning accuracy of the super-resolution imaging in the z direction is 100 nm or less, for example, 40 nm or less, for example, 20 nm or less; or, preferably, the super-resolution imaging
  • the full width at half maximum of the single molecule repeat positioning accuracy in the z direction is 250 nm or less, for example, 100 nm or less, for example, 50 nm or less.
  • the method for identifying a nucleic acid of the present disclosure can simplify the steps of nucleic acid recognition, efficiently label a target nucleic acid molecule, reduce non-specific binding of the probe to a nucleic acid other than the target nucleic acid, and improve the efficiency and accuracy of recognition, and particularly
  • the recognition of nucleic acids that facilitate shorter lengths and/or non-repetitive sequences is particularly suitable for obtaining super-resolution images of target nucleic acids.
  • the dilemma of selecting the hybridization temperature is eliminated, and the non-specific binding can be attenuated while increasing the specific hybridization intensity, thereby realizing a shorter (for example, less than 4.9 kb) target nucleic acid. High resolution imaging of fragments.
  • the nucleic acid recognition method of the present disclosure simplifies the operation steps and the requirements for the reagent, and does not require the user to establish an expensive and complicated nucleic acid coating production system, which not only saves cost, the nucleic acid coating and the probe quality are stably and controllable, but also operates. More convenient and feasible.
  • Figure 1a shows the structure of a target nucleic acid fragment inserted into the genome of the cell prepared in Example 1.
  • Fig. 1b shows the results of PCR detection of the cell sample (I) prepared in Example 1, showing that the positive sample contains the target nucleic acid fragment and the negative sample does not contain the target nucleic acid fragment.
  • Example 2 shows the sequence of the antisense strand of the 3.3 kb target nucleic acid fragment in Example 1.
  • Figure 3a shows fluorescence emission readings of hybridization probes MB1 to MB29 after co-reaction with corresponding complementary or non-complementary oligonucleotides at room temperature (22 °C).
  • Figure 3b shows fluorescence emission readings of hybridization probes MB1 to MB29 after co-incubation with corresponding complementary or non-complementary oligonucleotides at different temperatures.
  • Figure 3c shows the number of events recorded over time as they are imaged with different sample frame rates and laser power.
  • Figure 4a shows 1378 positional information obtained when STORM imaging was performed on the positive sample prepared in Example 4.
  • Figure 4b shows the distribution of the position information in Figure 4a in the x, y, z directions.
  • Figure 5 shows images of three exemplary target nucleic acids obtained after reconstitution with a conventional optical microscope and STORM super-resolution imaging for the positive samples prepared in Example 4.
  • FIG. 6 shows the intensity normalized distribution of the region where the red line is located in the three exemplary field images obtained by the STORM method in FIG. 5.
  • Fig. 7 shows the results of PCR detection of the cell sample (II) prepared in Example 7, showing that the positive sample contained the target nucleic acid fragment, and the negative sample contained no target nucleic acid fragment.
  • Figure 8 shows the sequence of the sense strand of the 2.5 kb target nucleic acid fragment in Example 7.
  • Figure 9a shows the fluorescence emission readings of hybridization probes MB30 to MB63 after co-reaction with corresponding complementary or non-complementary oligonucleotides at room temperature (22 °C).
  • Figure 9b shows the fluorescence emission readings of hybridization probes MB30 to MB63 after co-incubation with corresponding complementary or non-complementary oligonucleotides at different temperatures.
  • Figure 10 shows images of three exemplary target nucleic acids obtained after reconstitution of a positive sample prepared in Example 10 by conventional optical microscopy and STORM super-resolution imaging.
  • Fig. 11 is a view showing the normalized distribution of the intensity of the region where the red line is located in the three exemplary fields of view obtained by the STORM method in Fig. 10.
  • FIG. 12 shows a schematic diagram of a principle according to an exemplary embodiment of the present disclosure.
  • the present disclosure provides a method for identifying a nucleic acid, the method comprising the steps of:
  • the hybridization probe comprises a reporter group and the hybridization probe is in a different state in two cases to generate an identifiable signal by the reporter group: (i) the hybridization probe is at In the free state, (ii) the hybridization probe binds to the target nucleic acid.
  • the sample identified by the identification method of the present disclosure may be a natural or synthetic nucleic acid, or may be a tissue or a cell taken from an organism, a cultured cell, or the like.
  • the sample may be a tissue frozen section, a paraffin section, a cell slide, or the like. Tissue/cell lysate, etc.
  • Pretreatment of the sample can be carried out by reference to a pretreatment method of a conventional in situ hybridization sample.
  • the hybridization probe comprises a reporter group, and the hybridization probe differs in state under two conditions such that an identifiable signal is produced by the reporter group: (i) the hybridization probe is in a free state, ( Ii) the hybridization probe binds to the target nucleic acid.
  • the specific structure of the hybridization probe is not particularly limited.
  • a hybridization probe can comprise a binding region and a dissociation region, the dissociation region self-hybridizing when the hybridization probe is in a free state, the reporter group not emitting an identifiable signal; when the hybridization probe Upon specific binding of the binding region to a target sequence fragment of the target nucleic acid, the dissociation region dissociates and the reporter group emits an identifiable signal.
  • the relationship between the binding region and the dissociation region of the hybridization probe may be, for example, the dissociation region is included in the binding region, or the dissociation region partially overlaps the binding region, or the dissociation region is located outside the binding region.
  • the reporter group comprises a fluorophore and a quencher, the identifiable signal being fluorescent. That is, when the hybridization probe is in a free state, the dissociation region self-hybridizes, the fluorophore is quenched by the quenching group, so that the reporter group does not fluoresce; when the hybridization probe passes through When the binding region specifically binds to a target sequence fragment of the target nucleic acid, the dissociation region dissociates, and quenching of the fluorophore by the quenching group is released, whereby the reporter group fluoresces.
  • the hybridization probe may be a DNA probe, an RNA probe, a chemically modified oligonucleotide nucleic acid probe, a nucleic acid analog or the like.
  • the hybridization probe is a single stranded DNA probe or a single stranded RNA probe.
  • the hybridization probe has a stem-loop structure, such as a hairpin-like structure.
  • the hybridization probe comprises a loop structure that acts as a binding region, and at least two arm structures that serve as dissociation regions, wherein at least two of the arm structures are located on either side of the loop structure, when the hybridization probe is not in contact with the target nucleic acid,
  • the arm structure on the 5' side of the ring structure (hereinafter referred to as the 5' arm structure) and the arm structure on the 3' side of the ring structure (hereinafter referred to as the 3' arm structure) complementarily form a stable stem-like structure; the fluorophore and the quenching group respectively Located at the 5' end of the 5' arm structure and the 3' end of the 3' arm structure, and the positions of the fluorophore and the quenching group can be exchanged with each other, that is, the fluorophore can be quenched at the 5' end of the 5'
  • the length of the ring structure is not particularly limited as long as the ring structure has sufficient binding strength to the target nucleic acid at the hybridization temperature.
  • the length of the loop structure is preferably 20 to 60 nucleotides (i.e., 20 nt to 60 nt), more preferably 25 nt to 55 nt, and more preferably 30 nt to 50 nt, more preferably due to design difficulty, cost of preparing the probe, and the like. It is preferably 35 nt to 45 nt, and still more preferably 40 nt to 44 nt.
  • sequence identity between the loop structure and the reverse complement of the target nucleic acid is from 90% to 100%, preferably from 95% to 100%, preferably from 98% to 100%.
  • sequence identity between the loop structure and the reverse complement of the target nucleic acid is 100%, i.e., the loop structure is inversely complementary to the target nucleic acid.
  • the length of the arm structure and the nucleic acid sequence are not particularly limited, and the design of the arm structure can be carried out by referring to the method described in Non-Patent Document 4, which is incorporated herein by reference.
  • the arm structure has a length of from 4 nt to 10 nt, preferably from 5 nt to 9 nt, preferably from 6 nt to 8 nt, more preferably 7 nt.
  • the hybridization probe may be a single-stranded nucleic acid represented by the following general formula (1):
  • X 1 to X m , Y 1 to Y n , and X′ 1 to X′ m represent arbitrary nucleotides
  • the nucleotide chain fragment represented by Y 1 Y 2 ... Y n is inversely complementary to the target nucleic acid
  • a nucleotide chain fragment represented by X 1 X 2 ... X m is inversely complementary to a nucleotide strand fragment represented by X' 1 X' 2 ... X' m ,
  • the nucleotide chain fragment represented by Y 1 Y 2 ... Y n binds to the target nucleic acid with a T m value higher than that of the nucleotide chain fragment represented by X 1 X 2 ... X m and by X' 1 X' 2 ... X ' m represents the T m value of the nucleotide chain fragment binding,
  • the nucleotide X 1 is conjugated with a fluorophore and the nucleotide X' m is conjugated with a quenching group, or the nucleotide X 1 is conjugated with a quenching group and the nucleotide X' m is conjugated thereto.
  • Fluorophore
  • n is an integer selected from the group consisting of 20 to 60, preferably 25 to 55, preferably 30 to 50, preferably 35 to 45, more preferably 40 to 44.
  • the nucleotide chain fragment represented by Y 1 Y 2 ... Y n satisfies at least one of the following conditions: i) a T m value of not lower than 70 ° C, ii) when the sample contains a genomic nucleic acid, by Y 1
  • the sequence of the nucleotide chain fragment represented by Y 2 ... Y n is not more than 25 nt in length from the genomic non-target nucleic acid contained in the sample, iii) does not contain 6 or more consecutive repeating nucleotides, iv) There is no secondary structure formation at temperatures equal to or higher than the hybridization temperature.
  • the nucleotide chain fragment represented by X 1 X 2 ... X m and the nucleotide chain fragment represented by X' 1 X' 2 ... X' m satisfy at least one of the following conditions: i) by X 1
  • the nucleotide chain fragment represented by X 2 ... X m and the nucleotide chain fragment represented by X' 1 X' 2 ... X' m are high GC content fragments (75% to 100%), ii) by X 1 X 2 ... T m values of X m represents a fragment of a nucleotide chain bound to the nucleotide chain fragment consisting of X '1 X' 2 ...
  • X 'm in the range indicated 50 °C ⁇ 60 °C of, iii) represented by X 1
  • the nucleotide chain fragment represented by X 2 ... X m and the nucleotide chain fragment represented by X' 1 X' 2 ... X' m are not formed with the nucleotide chain fragment represented by Y 1 Y 2 ... Y n secondary structure.
  • the base in the conjugated fluorophore is C (cytosine).
  • the hybridization probe is a molecular beacon.
  • the reporter group of the hybridization probe is an identifiable label, preferably a fluorophore and a quencher.
  • the maximum emission wavelength of the fluorophore is close to the maximum absorption wavelength of the quenching mass to achieve an optimized quenching effect.
  • Preferred fluorophores are, for example, by Thermo Fisher as Molecular The Alexa Fluor series of dyes sold under the trade name.
  • quenching groups for use in accordance with the present disclosure include, but are not limited to, 4-(4'-dimethylaminoazophenyl)benzoic acid (DABCYL), black hole quencher-1 (black hole quencher 1, BHQ-1), black hole quencher-2 (BHQ-2), black hole quencher-3 (BHQ-3), and the like.
  • DBCYL 4-(4'-dimethylaminoazophenyl)benzoic acid
  • black hole quencher-1 black hole quencher 1, BHQ-1
  • black hole quencher-2 BHQ-2
  • BHQ-3 black hole quencher-3
  • the target nucleic acid is the nucleic acid or nucleic acid fragment to be recognized by the methods of the present disclosure.
  • the target nucleic acid can be, for example, single or double stranded DNA, including but not limited to genomic DNA, cDNA, etc., or single or double stranded RNA, including but not limited to messenger RNA, ribosomal RNA, microRNA, viral RNA, and the like.
  • the length of the target nucleic acid is less than 4.9 kb; more preferably, the length of the target nucleic acid is 3.3 kb or less, further preferably 2.5 kb or less, and particularly preferably 2.0 kb to 2.5 kb.
  • the target nucleic acid does not comprise a repeat sequence.
  • the target nucleic acid contains an enhancer sequence or a promoter sequence.
  • a repeat sequence is defined as the same unit or symmetry fragment that occurs multiple times in different positions in the genome or in the same large range of positions, including but not limited to mild, moderate, highly repetitive sequences such as ALU family, centromere , telomere, etc.; a non-repetitive sequence is defined as a sequence unique to the entire genome, and there are no repeats similar to it (in polyploids, such as diploids, non-repetitive sequences can appear in the paired chromosomes, respectively) .
  • the advantages of the present disclosure become more apparent if used to identify target nucleic acids that do not contain repeat sequences.
  • the length of the target nucleic acid containing no repeat sequence is short, for example, less than 4.9 kb
  • the total length of the hybridization probe bound to the target nucleic acid is limited by the length of the target nucleic acid, the sequence characteristics, and the length of the nucleic acid sequence of each hybrid probe binding region.
  • the number of reporter groups on the hybrid probes that successfully bind to the target nucleic acid is small, the total signal is weak, and it is difficult to distinguish from the background, which hinders the further improvement of the recognition accuracy. While the nucleic acid recognition method of the present disclosure successfully achieves high resolution imaging of short fragments of non-repetitive target nucleic acids less than 4.9 kb in length, as exemplified in Examples 6 and 11 below.
  • the method and conditions for bringing the sample into contact with the hybridization probe are not particularly limited as long as the hybridization probe is bound to a target nucleic acid which may be present in the sample.
  • the sample may be co-incubated with the self-quenching probe at a temperature of from 70 ° C to 80 ° C, preferably from 73 ° C to 77 ° C, more preferably 75 ° C, and then from 10 ° C to 42 ° C, more preferably from 12 ° C to 38 °
  • Hybridization is carried out at a temperature of °C, more preferably 14 ° C to 34 ° C, more preferably 16 ° C to 34 ° C, still more preferably 18 ° C to 26 ° C, still more preferably 22 ° C.
  • the manner of identifying whether or not the self-quenching probe binds to the target nucleic acid can be appropriately selected depending on the characteristics of the sample, the self-quenching probe and the target nucleic acid to be detected, in accordance with the purpose of recognition.
  • it can be read by a fluorescence spectrophotometer or by fluorescence microscopy, such as ordinary fluorescence microscopy, confocal microscopy, or fluorescence super-resolution imaging.
  • the reporter group includes a fluorophore and a quencher
  • the wavelength at which the fluorescence is excited and recognized can be appropriately selected depending on the nature of the fluorophore itself.
  • the imaging resolution achieved by the nucleic acid recognition method of the present disclosure reaches 50 nm or less in the xy direction, particularly 9 nm, and/or in the standard deviation of single molecule repeat positioning accuracy.
  • This is a high-resolution imaging that is difficult to achieve in the prior art, especially for short segments of non-repetitive target nucleic acids less than 4.9 kb in length, and no high-resolution imaging has been reported in the prior art.
  • the laser power used for the excitation can be appropriately adjusted depending on the state of the sample, the type of the fluorophore, and the like.
  • the laser power used in the excitation is from 0.80 kW/cm 2 to 2.20 kW/cm 2 .
  • the laser power used in the excitation is 0.80 kW/cm 2 to 2.00 kW/cm 2 , more preferably 0.90 kW/cm 2 to 1.45 kW/cm 2 , further preferably 1.00 kW/cm 2 . .
  • the fluorophore can be excited to obtain a sufficiently strong fluorescent signal, and on the other hand, to delay the progress of photobleaching of the fluorophore.
  • the sampling frequency used for identifying the fluorescence can be appropriately adjusted according to conditions such as the demand for image quality, the performance of the image forming apparatus, and the like.
  • the sampling frame frequency used for identifying the fluorescence is preferably 10 Hz or more, more preferably 50 Hz or more, still more preferably 60 Hz or more, still more preferably 85 Hz or more.
  • Human SK-N-SH cells were infected with the engineered lLL3.7-based lentiviral vector. After infection, cells positive for EGFP expression were screened by flow cytometry (BD FACSAria SORP Cell Sorter). In MEM medium (Gibco) containing 10% fetal bovine serum, coverslip (Fisherbrand TM Coverglass for Growth TM Cover Glasses, Num 12-545-82) culturing the EGFP positive cells (positive cells) or Human SK-N-SH cells (negative control) that were not infected with lentivirus.
  • MEM medium Gibco
  • coverslip Fisherbrand TM Coverglass for Growth TM Cover Glasses, Num 12-545-82
  • a viral RNA fragment of about 3.3 kb in length of the above lentiviral vector was reverse transcribed into the genome of SK-N-SH cells.
  • the integrated 3.3 kb fragment (SEQ ID NO: 30) contains the sequence encoding EGFP that is initiated by the CMV promoter.
  • a segment of the 3.3 kb fragment of about 2.5 kb in length is exemplified as an exemplary target nucleic acid fragment, which is identified in the recognition method according to the present disclosure as detailed below (Fig. 1a).
  • the target nucleic acid fragment is contained in the genome of the cell as the positive sample identified, and there is only one copy of the target nucleic acid fragment at each integration site.
  • the cells that are the negative samples identified do not contain the above target nucleic acid fragments. The above characteristics of the positive and negative samples were confirmed by PCR (Fig. 1b).
  • An exemplary hybridization probe provided in this example is a 56 nt single stranded oligonucleotide comprising a 42 nt loop structure and a 7 nt arm structure flanking the loop structure.
  • One end of the hybridization probe is conjugated to a fluorophore-like Alexa-647, and the other end is conjugated to a quenching group of BHQ3.
  • Twenty-nine hybridization probes designated MB1 to MB29, were designed and synthesized by Life Technology. The sequences of these 29 hybridization probes are described in the sequence listing (SEQ ID NOS: 1 to 29).
  • the sequence of the loop structure is inversely complementary to a portion of the antisense strand of the target nucleic acid fragment of Example 1 (ie, identical to a portion of the sense strand of the target nucleic acid fragment), and 29 hybridization probes can pass through their respective loop structures
  • the target nucleic acid fragment is labeled along the strand of the target nucleic acid.
  • the sequences of the two arm structures in each probe are complementary to each other in the opposite direction.
  • the fluorescence of Alexa-647 is quenched by BHQ3; when the hybridization probe binds to the target nucleic acid fragment, the probe's Alexa-647 can be excited to emit fluorescence and the fluorescence is not BHQ3 impact.
  • the sequence of the antisense strand of the 3.3 kb target nucleic acid fragment in Example 1 is shown in Figure 2, and the sequence fragment targeted by the hybridization probes MB1 to MB29 in the target nucleic acid fragment is underlined. It can be seen that 29 hybridization probes can label non-repetitive sequences of target nucleic acid fragments.
  • 29 sequences as hybridization probes T m value of the ring structure, an arm structure T m values in Table 1.
  • the ring structure is represented by uppercase letters and the arm structure is represented by lowercase letters.
  • hybridization probe the oligonucleotide complementary to the hybridization probe (CS), and the oligonucleoside not complementary to the hybridization probe sequence are dissolved in a buffer containing 50 mM NaCl, 1 mM EDTA, and 10 mM Tris (pH 7.4). Acid (NCS).
  • 80 nM hybridization probes (MB1 to MB29) were reacted with the corresponding CS (1600 nM) or NCS (1600 nM) in a 2 x SSC, 50% formamide hybridization solution for 30 min at room temperature. Thereafter, a fluorescence emission reading was read at 665 nm using a fluorescence spectrophotometer with a laser of 647 nm. The readings for each hybridization probe are shown in Figure 3a. For each hybridization probe, the fluorescence emission readings after co-reaction with CS were significantly higher than the fluorescence emission readings after co-reaction with NCS, indicating specific binding of each hybridization probe to the target sequence.
  • 80nM hybridization probes were reacted with the corresponding CS (1600nM) or NCS (1600nM) for 30min at different temperatures (42°C, 38°C, 34°C, 30°C, 26°C, 22°C, 18°C, 14°C). . Thereafter, the fluorescence emission reading was read at 665 nm by excitation with a laser of 647 nm. The results are shown in Figure 3b.
  • the cells on the coverslips of Example 1 were grown to 80% confluence, fixed in 4% paraformaldehyde-PBS for 10 min, infiltrated in PBS for 2 min, treated with 1 mg/mL sodium borohydride for 7 min, and infiltrated with ddH2O for 2 min.
  • the cells were then immersed in 25% glycerol-PBS for 40-50 min, frozen in liquid nitrogen, thawed, and the freeze-thaw cycle was repeated 3 times.
  • the cells were then treated with RNase A (100 ⁇ g/ml) for 1 hr at 37 °C. After washing with PBS, the cells were infiltrated with PBS for 5 minutes.
  • the cells were pre-warmed in 2X SSC buffer at 75 °C for 5 min, then 2 x SSC buffer was exchanged for 2 x SSC buffer, 80% deionized formamide and treatment was continued at 75 °C for 3 min. The cells were then treated with cold ethanol infiltration (concentration 75%, 90%, 100%) for 2 min each. The cells were blocked overnight at room temperature (22 °C) with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 x SSC buffer. The cells were incubated with the newly configured 1.5% FISH blocking buffer (Roche), 50% formamide and 2 x SSC buffer at 42 °C until the time of hybridization, 3 to 6 hours after reaction with the hybridization probe.
  • a total concentration of 714 nM of the hybridization probe mixture synthesized in Example 2 (containing all probes), 1.5% FISH blocking buffer, 50% formamide and 2 x SSC buffer was prepared using 14 ⁇ L of the mixture and each glass.
  • the cells on the slide were hybridized at 22 ° C for 16-20 hours.
  • the cells were washed with 50% formamide and 2 x SSC buffer for 40-50 min at room temperature and fixed with 4% paraformaldehyde-PBS for 5-10 min.
  • the obtained positive and negative test samples were infiltrated in 0.25 x SSC buffer at 4 °C.
  • Example 4 The sample obtained in Example 4 was identified by the following method. Imaging was performed by random optical reconstruction microscopy (STORM) using an inverted optical microscope (IX-71, Olympus) equipped with a 100x oil immersion objective (UPlanSApo, N.A. 1.40, Olympus). For the labeled Alexa-647 on the hybridization probe, Alexa647 state radiant fluorescence was excited with a 641 nm laser and Alexa-647 was converted to a dark state and reactivated with a 405 nm laser. Each transition from a bright state to a dark state is recorded as an on/off event.
  • ERP random optical reconstruction microscopy
  • IX-71 inverted optical microscope
  • UPlanSApo 100x oil immersion objective
  • the hybridization probe marks a non-repetitive sequence of the target nucleic acid, the label density is relatively sparse, and the recognition of the fluorescence may be interfered by the autofluorescence of the cell.
  • the laser power and sampling frame rate were adjusted, and multiple sets of experiments were performed.
  • the specific conditions of each group are shown in Table 2.
  • the false positive rate (FDR) is recorded as the ratio of the number of on/off events recorded from the negative sample to the number of on/off events recorded from the positive sample under the same conditions.
  • the average FDR under each condition is also shown in Table 2.
  • the average false positive rate of conditions IX, X, XI, and XII is relatively low, which is a superior condition for STORM imaging using the hybridization probe of the present disclosure.
  • Example 4 The positive samples prepared in Example 4 were subjected to STORM imaging under Condition XII of Example 5. By performing light-dark conversion on Alexa-647, a cluster distribution formed by repeated positioning of a plurality of fluorescent single molecules is obtained. The 1378 localization distribution accumulated from 53 clusters is shown in Fig. 4a. The repeated positioning satisfies the Gaussian distribution, with a full width at half maximum of 22 nm in the lateral direction and 52 nm in the axial direction, indicating that a resolution of about 20 to 30 nm is obtained in the xy direction and a resolution of about 50 to 60 nm is obtained in the z direction ( Figure 4b).
  • FIG. 5 shows an image of an exemplary field of view of 3 cell nuclei.
  • columns 1, 2, and 3 are images obtained by conventional optical microscopy, and it can be seen that the fluorescent spots of the labeled target nucleic acid cannot be discerned.
  • Column 4 is the super-resolution color image obtained by STORM imaging method in the green box area of columns 1, 2 and 3.
  • Column 5 is the enlargement of the white box area in the image of column 4, which more clearly shows the target nucleic acid in the super-resolution image. The morphology and the number of normal molecules collected by the target nucleic acid during imaging in the lower right corner.
  • the color of the super-resolution color image represents single-molecule localization in the z-direction (-350 to 350 nm).
  • Figure 6 is a graph showing the normalized distribution of the intensity of the region (i, ii, iii) of the red line in the target nucleic acid super-resolution structure obtained by the STORM imaging method in Figure 5, showing that the imaging accuracy can resolve the micro-distance of 44 nm in the structure.
  • a 3 kb fragment (mm9 mouse whole genome coordinate site, Chr6: 122612566-122615608, SEQ ID NO: 66) was knocked out in mouse CJ9 stem cells using CRISPR/Cas9 gene editing technology. After screening, homozygous knock was obtained. The cells were used as a negative control.
  • MES in complete medium, the cover glass (Fisherbrand TM Coverglass for Growth TM Cover Glasses, Num 12-545-82) culturing wild-type CJ9 stem cells (positive cells) by the above-described mouse or CRISPR / Cas9 obtained on a purely technical The zygote knockout cells (negative control).
  • a segment of chromosome 6 in the genome of about 2.5 kb in length (mm9 mouse whole genome coordinate site, Chr6: 122612623-122615179) as an exemplary target nucleic acid fragment (SEQ ID NO: 65), It is identified in the detailed identification method according to the present disclosure described below.
  • the target nucleic acid fragment is contained in the cell genome as the recognized positive sample, and only one copy of the target nucleic acid fragment is present on chromosome 6 in the entire genome.
  • the cells that are the negative samples identified do not contain the above target nucleic acid fragments, since a 3.3 kb nucleic acid fragment containing a 2.5 kb target nucleic acid has been knocked out by the CRISPR/Cas9 gene editing technique.
  • the above characteristics of the positive and negative samples were confirmed by PCR (Fig. 7).
  • An exemplary hybridization probe provided in this example is a 56 nt single stranded oligonucleotide comprising a 42 nt loop structure and a 7 nt arm structure flanking the loop structure.
  • One end of the hybridization probe is conjugated to a fluorophore-like Alexa-647, and the other end is conjugated to a quenching group of BHQ3.
  • hybridization probes designated MB30 to MB63, were designed and synthesized by Life Technology. The sequences of these 34 hybridization probes are described in the sequence listing (SEQ ID NOS: 31-64). Among them, 24 hybridization probes (MB30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, 47, 49, 51, 52, 53, 55, 56, 58, 59, The sequence of the loop structure of 61, 62, 63) is inversely complementary to a portion of the antisense strand of the target nucleic acid fragment of Example 7 (ie, identical to a portion of the sense strand of the target nucleic acid fragment), and the remaining 10 hybrids The sequence of the loop structure of the needle (MB32, 34, 40, 43, 46, 48, 50, 54, 57, 60) is inversely complementary to a portion of the sense strand of the target nucleic acid fragment of Example 7 (ie, with the target A portion of the antisense strand
  • These 34 hybridization probes can label the target nucleic acid fragment along the strand of the target nucleic acid by their respective loop structures.
  • the sequences of the two arm structures in each probe are complementary to each other in the opposite direction. Therefore, when the hybridization probe does not bind to the target nucleic acid fragment, the fluorescence of Alexa-647 is quenched by BHQ3; when the hybridization probe binds to the target nucleic acid fragment, the probe's Alexa-647 can be excited to emit fluorescence and the fluorescence is not BHQ3 impact.
  • the sequence of the sense strand of the 2.5 kb target nucleic acid fragment in Example 7 is shown in Figure 8, and the sequence fragment targeted by the hybridization probes MB30 to MB63 in the target nucleic acid fragment is underlined or bolded. Wherein, the underline indicates that the hybridization probe targets the antisense strand of the target nucleic acid, and bold indicates that the hybridization probe targets the sense strand of the target nucleic acid. It can be seen that 34 hybridization probes can label non-repetitive sequences of target nucleic acid fragments.
  • hybridization probe the oligonucleotide complementary to the hybridization probe (CS), and the oligonucleoside not complementary to the hybridization probe sequence are dissolved in a buffer containing 50 mM NaCl, 1 mM EDTA, and 10 mM Tris (pH 7.4). Acid (NCS).
  • 80 nM hybridization probes (MB30 to MB63) were reacted with the corresponding CS (1600 nM) or NCS (1600 nM) in a 2 x SSC, 50% formamide hybridization solution for 30 min at room temperature. Thereafter, a fluorescence emission reading was read at 665 nm using a fluorescence spectrophotometer with a laser of 647 nm. The readings for each hybridization probe are shown in Figure 9a. For each hybridization probe, the fluorescence emission readings after co-reaction with CS were significantly higher than the fluorescence emission readings after co-reaction with NCS, indicating specific binding of each hybridization probe to the target sequence.
  • the cells on the coverslips of Example 7 were grown to 80% confluence, fixed in 4% paraformaldehyde-PBS for 10 min, infiltrated in PBS for 2 min, treated with 1 mg/mL sodium borohydride for 7 min, and infiltrated with ddH2O for 2 min.
  • the cells were then immersed in 25% glycerol-PBS for 40-50 min, frozen in liquid nitrogen, thawed, and the freeze-thaw cycle was repeated 3 times.
  • the cells were then treated with RNase A (100 ⁇ g/ml) for 1 hr at 37 °C. After washing with PBS, the cells were infiltrated with PBS for 5 minutes.
  • the cells were pre-warmed in 2 x SSC buffer at 75 °C for 5 min, then 2 x SSC buffer was exchanged for 2 x SSC buffer, 80% deionized formamide and treatment was continued at 75 °C for 3 min. The cells were then treated with cold ethanol infiltration (concentration 75%, 90%, 100%) for 2 min each. The cells were blocked overnight at room temperature (22 °C) with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 x SSC buffer. The cells were incubated with the newly configured 1.5% FISH blocking buffer (Roche), 50% formamide and 2 x SSC buffer at 42 °C until the time of hybridization, 3 to 6 hours after reaction with the hybridization probe.
  • a total concentration of 714 nM of the hybridization probe mixture synthesized in Example 2 (containing all probes), 1.5% FISH blocking buffer, 50% formamide and 2 x SSC buffer was prepared using 14 ⁇ L of the mixture and each glass.
  • the cells on the slide were hybridized at 22 ° C for 16-20 hours.
  • the cells were washed with 50% formamide and 2 x SSC buffer for 40-50 min at room temperature and fixed with 4% paraformaldehyde-PBS for 5-10 min.
  • the obtained positive and negative test samples were infiltrated in 0.25 x SSC buffer at 4 °C.
  • Example 10 The positive sample prepared in Example 10 was subjected to STORM imaging under Condition XII of Example 5. By performing light-dark conversion on Alexa-647, a cluster distribution formed by repeated positioning of a plurality of fluorescent single molecules is obtained.
  • FIG. 10 shows an image of an exemplary field of view of 3 cell nuclei.
  • columns 1, 2, and 3 are images obtained by conventional optical microscopy, and it can be seen that the fluorescent spots of the labeled target nucleic acid cannot be discerned.
  • Column 4 is the super-resolution color image obtained by STORM imaging method in the green box area of columns 1, 2 and 3.
  • Column 5 is the enlargement of the white box area in the image of column 4, which more clearly shows the target nucleic acid in the super-resolution image. The morphology and the number of normal molecules collected by the target nucleic acid during imaging in the lower right corner.
  • the color of the super-resolution color image represents single-molecule localization in the z-direction (-350 to 350 nm).
  • Figure 11 is a graph showing the normalized distribution of the intensity of the region (i, ii, iii) of the red line in the target nucleic acid super-resolution structure obtained by the STORM imaging method in Figure 10, showing that the imaging accuracy can resolve the distance between the structures by 58 nm (i And a microstructure of 37 nm (ii) and a separate structure of 25-34 nm (iii). It can be seen that the method of the present disclosure has particular advantages for super-resolution imaging applications.

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Abstract

La présente invention concerne un procédé de reconnaissance pour acide nucléique. Le procédé de reconnaissance comprend les étapes suivantes : a. établir le contact d'un échantillon avec une sonde d'hybridation ; b. éventuellement éluer la sonde d'hybridation qui n'est pas liée à un acide nucléique cible ; et c. reconnaître si la sonde d'hybridation est liée à l'acide nucléique cible, où la sonde d'hybridation comprend des groupes rapporteurs, et la sonde d'hybridation, de sorte que des signaux reconnaissables sont produits par les groupes rapporteurs, présente différents états sous les deux conditions suivantes : (i) la sonde d'hybridation se trouve en un état libre, et (ii) la sonde d'hybridation est liée à l'acide nucléique cible. La présente invention concerne également l'utilisation de la sonde d'hybridation dans l'imagerie de microscopie par fluorescence de l'acide nucléique cible. Le procédé de reconnaissance peut simplifier les étapes de fonctionnement pour la reconnaissance de l'acide nucléique, marquer efficacement les molécules d'acide nucléique cible, réduire la liaison non spécifique de la sonde aux acides nucléiques autres que l'acide nucléique cible, améliorer l'efficacité et la précision de reconnaissance, et particulièrement tirer parti de la reconnaissance d'acides nucléiques présentant une plus petite longueur et/ou des séquences non répétitives, et convient pour acquérir des images en super-résolution de l'acide nucléique cible.
PCT/CN2018/083921 2017-04-24 2018-04-20 Procédé de reconnaissance précise pour acide nucléique WO2018196691A1 (fr)

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