KR20230152975A - Ligation-free isothermal nucleic acid amplification for detection of target nucleic acid - Google Patents

Abstract

The present invention relates to an isothermal nucleic acid amplification method using a chain displacement nucleic acid polymerase and two primers designed to bind adjacent to a target nucleic acid, including a first probe having a hairpin structure and a nucleic acid polymerization product designed to have a hairpin structure. Using a probe set including a second probe, the target nucleic acid can be amplified even at isothermal temperature by repeatedly self-priming the amplification product of the target nucleic acid. Unlike the prior art, a separate ligase and temperature control device is not required, and target nucleic acids can be amplified and detected multiple times with high selectivity and sensitivity, so it can be useful for actual disease diagnosis based on this. .

Description

Isothermal nucleic acid amplification method for detecting target nucleic acid without ligation {Ligation-free isothermal nucleic acid amplification for detection of target nucleic acid}

The present invention relates to a technology for amplifying nucleic acids at isotherm without a ligation reaction and a method for detecting target nucleic acids based on this.

PCR (polymerase chain reaction), a conventional nucleic acid amplification technology, can analyze multiple samples at the same time and even analyze the genotype of the virus, but requires DNA denaturation, primer annealing, and nucleic acid extension by polymerase. ) A thermocycler is required to change the temperature required for the step, precision is reduced because it relies only on hybridization of primers to target nucleic acids, and the analysis cost increases and analysis time becomes longer when detecting multiple target nucleic acids. there is.

Ligation-dependent nucleic acid amplification, which increases precision, ligates the two probes with ligase when two probes designed to bind adjacent to the target nucleic acid hybridize to the target nucleic acid. This is a method of amplification with the above two types of probes connected. The ligation-dependent nucleic acid amplification method described above has the advantage of being able to detect the target nucleic acid very selectively because ligation can proceed only when the two types of probes are completely matched to the target nucleic acid. An additional enzymatic ligation agent called ligase is required, and in order to amplify the bound probe, a PCR primer binding sequence is additionally required at the ends of the above two types of probes, and expensive thermal cycling equipment for amplification is still required. Since it requires a delay time to reach the nucleic acid denaturation temperature, it has the disadvantage of requiring a long time until the amplification reaction is completed.

Meanwhile, as the demand for the development of point-of-care testing (POCT) technology has recently increased, the shortcomings of the existing polymerase chain reaction (PCR) technology, which requires an expensive temperature control device, have been resolved and the device has been improved. There is growing interest in the development of alternative technologies that can achieve miniaturization. The technology that overcomes these problems and allows it to be applied to point-of-care diagnostic systems is the isothermal nucleic acid amplification method. Isothermal nucleic acid amplification is similar to conventional PCR, but the amplification reaction is performed at a constant temperature without temperature changes required for DNA denaturation, primer annealing, and nucleic acid extension by polymerase in the PCR process. It is a technique that can be performed. In line with this technology trend, since the early 1990s, nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HAD), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), and loop-mediated isothermal (LAMP) Various isothermal nucleic acid amplification methods, such as amplification, RCA (rolling circle amplification), and EXPAR (exponential amplification reaction), have been actively developed.

However, in these conventional PCR or isothermal nucleic acid amplification methods, a primer-duplex binding phenomenon occurs during the amplification process, which is amplification by binding between primers using an excess amount, resulting in the amplification of non-specific nucleic acids and the precision of nucleic acid amplification is impaired. In particular, the problem is very serious in isothermal nucleic acid amplification methods that use primers that are relatively long compared to PCR primers. Therefore, because of these problems, multiplex amplification using various types of primers is difficult in the isothermal nucleic acid amplification method. Therefore, there is a need for an isothermal nucleic acid amplification technology that allows multiple amplification and detection of target nucleic acids while improving sensitivity.

One object of the present invention is to provide a probe set capable of isothermally amplifying a target nucleic acid without a ligation reaction and a composition for amplifying a target nucleic acid using the same.

In addition, another object of the present invention is to provide a kit for detecting a target nucleic acid that can amplify the target nucleic acid at isotherm without a ligation reaction.

In addition, another object of the present invention is to provide a method of isothermally amplifying a target nucleic acid without a ligation reaction and a method of detecting the target nucleic acid thereby.

In order to achieve the above object, one aspect of the present invention includes a first base sequence complementary to the first region of the target nucleic acid, and a base sequence forming a hairpin structure linked to the 3′-end of the first base sequence. first probe; And a second probe comprising a second base sequence complementary to the second region of the target nucleic acid, and a base sequence encoding a hairpin structure linked to the 5′-end of the second base sequence, and comprising the hairpin structure. A probe set is provided in which the encoding base sequence is modified to reduce base overlap interactions.

In addition, in order to achieve the above object, another aspect of the present invention provides a composition for amplifying a target nucleic acid comprising the probe set and a chain displacement nucleic acid polymerase.

In addition, in order to achieve the above object, another aspect of the present invention is a composition for amplifying the target nucleic acid; gRNA that binds to at least a portion of the target nucleic acid; Cas12 enzyme; and a single-stranded nucleic acid probe containing a fluorescent dye and a quenching agent.

Additionally, in order to achieve the above object, another aspect of the present invention provides a method of amplifying a target nucleic acid using the composition for amplifying the target nucleic acid.

In addition, in order to achieve the above object, another aspect of the present invention includes the steps of amplifying a target nucleic acid using the composition for amplifying the target nucleic acid; Generating a fluorescent signal using a single-stranded nucleic acid probe containing gRNA, Cas12, a fluorescent dye, and a quencher that binds to at least a portion of the target nucleic acid.

In the case of the primer set of the present invention, the base sequence encoding the hairpin structure included in the first probe and the hairpin structure included in the second probe modified to reduce base overlapping interactions is itself a hairpin structure when the target nucleic acid is extended. can be formed, and the self-formed hairpin structure can act as a primer (self-priming). Therefore, by using the probe set of the present invention, there is an effect of amplifying the target nucleic acid at isotherm, without the need to adjust the temperature for each step of the reaction like in PCR.

In addition, with the probe set of the present invention, by using a nucleic acid polymerase that exhibits chain displacement even when there is a gap in the template, such as Bst polymerase, the first and second probes included in the probe set are Even if it is not gated, it has the effect of specifically amplifying the target nucleic acid.

Ultimately, by using the probe set of the present invention and the composition for target nucleic acid amplification containing the same, the target nucleic acid can be amplified isothermally without the use of additional ligation agents. In addition, since the fluorescent signal for the target nucleic acid can be amplified using the amplification method of the present invention and the trans-cleavage activity of CRISPR/Cas12, there is an advantage in that the target nucleic acid can be detected with high selectivity and sensitivity. Based on these characteristics, it can be useful for body fluid biopsies that do not require invasive procedures, and can also be used for molecular-level diagnosis of diseases. The present invention can be usefully used as a platform for various biosensors, and has the advantage of being able to analyze multiple target nucleic acids, so it can be usefully used for disease prevention, early diagnosis, and development and prescription of personalized medicine through analysis of various target nucleic acids.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figure 1 is a schematic diagram schematically showing the target nucleic acid detection method based on the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 2 shows the evaluation of the ligation-independent nucleic acid amplification reaction of the present invention. Figure 2(a) shows the real-time fluorescence response of the target nucleic acid under various component conditions depending on the presence or absence of the target nucleic acid (ERBB2 RNA), ligase, and DNA polymerase, and Figure 2(b) shows the real-time fluorescence response of the nucleic acid amplification on agar. This is the result of electrophoresis using Ross gel.
Figure 3 shows the real-time fluorescence response of the target nucleic acid under various component conditions depending on the presence or absence of the target nucleic acid (ERBB2 RNA), the first probe (PS probe), and the second probe (FW probe).
Figure 4 is an agarose gel electrophoresis result confirming that an intermediate product of the ligation-independent nucleic acid amplification reaction of the present invention was produced.
Figure 5 shows results showing that, unlike a general second probe (PS probe), a nucleic acid amplification reaction does not occur when a second probe (PS probe) whose 3'-end is blocked with biotin is used.
Figure 6 shows the results of comparing amplification efficiency according to the length (0, 12, 20, and 25) of the PS sequence in the second probe (PS probe) in order to optimize the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 7 shows the results of comparing amplification efficiency according to the amplification reaction temperature (50-65°C) in order to optimize the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 8 shows the results of comparing the amplification efficiency between DNA polymerases to optimize the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 9 shows the results of comparing the amplification efficiency of each concentration (5-40U) of Bst DNA polymerase to optimize the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 10 shows the results of comparing amplification efficiency by probe concentration (1-100 nM) to optimize the ligation-independent nucleic acid amplification reaction of the present invention.
Figure 11 compares the results of gene amplification reactions according to the distance between the first probe and the second probe in the target nucleic acid or the number of mismatch sequences between the first probe and the target nucleic acid.
Figure 12 is a result showing the sensitivity of the ligation-independent isothermal nucleic acid amplification reaction of the present invention. Figure 12(a)(b) is the real-time fluorescence reaction result at various target RNA concentrations, and Figure 12(c) is the result of the real-time fluorescence reaction at various target RNA concentrations. It is a function graph showing the fluorescence intensity, and Figure 12(d) shows the results of real-time fluorescence measurement of target nucleic acids in the presence of various non-target nucleic acids.
Figure 13 shows results showing that simultaneous detection of target nucleic acids is possible in a nucleic acid mixture containing non-target nucleic acids.
Figure 14 is a result showing that simultaneous detection of target genes is possible even in actual cells. Figure 14(a) is a schematic diagram of mRNA extraction from a cell line using the ligation-independent isothermal nucleic acid amplification method of the present invention, and (b) The real-time fluorescence response of ERB2 and GRB7 from HCC1143 and HCC1954 cell lines using a gation-independent isothermal nucleic acid amplification reaction and the fluorescence intensity for 30 minutes for each cell line are shown. Figure 14(c) shows the results of analyzing ERBB2 and GRB7 genes by qRT-PCR in HCC1143 and HCC1954 cell lines (n = 3, error bar = standard deviation).
Figure 15 is a result showing that simultaneous detection of target genes is possible in tissues and body fluids of actual animal models. Figure 15(a) shows mRNA from tumor-bearing mouse tissue samples and urine samples using a ligation-independent nucleic acid amplification reaction. This is a schematic diagram of extraction. (b) A photograph of a tumor-bearing mouse and a normal mouse. Figure 15(c)(d) shows real-time fluorescence reaction results for ERBB2 and GRB7 genes in (c) tissue and (d) urine samples using ligation-independent nucleic acid amplification reaction and fluorescence intensity for 30 minutes in mouse model. (n = 3, error bars = standard deviation).
Figure 16 shows the results of analyzing ERBB2 and GRB7 genes by qRT-PCR in tissue samples extracted from a tumor mouse model.

First, the terms used in the present invention are defined.

In the present invention, the term 'ligation' includes any method of linking two nucleic acid fragments by a covalent bond. The internucleotide linkages between the nucleic acid segments created by ligation include phosphodiester bonds and other linkages. The ligation may be a phosphodiester bond by an enzymatic ligation agent, such as a ligase. The ligase is an enzymatic ligation agent, including SplintR ligase, bacteriophage T4 ligase, E.coli ligase, Afu ligase, Taq ligase, Tfl ligase, Mth ligase, Tth ligase, and Tth HB8 ligase. , Thermus species AK16D ligase, Ape ligase, LigTk ligase, Aae ligase, Rm ligase, Pfu ligase, ribozyme, and variants thereof. Additionally, the ligation agent may include ligation using a non-enzymatic ligation agent or photoligation using light of a suitable wavelength. Non-enzymatic methods for ligation can form linkages between different nucleotides. The other linkages between nucleotides include a thiophosphorylacetylamino group formed between an α-haloacyl group and a phosphothioate group, a 5'-phosphorothioester formed between a phosphothioate group and a tosylate group or an iodide group. , and the formation of covalent bonds between suitable reactive groups such as pyrophosphate linkages.

In the present invention, the term 'ligation-dependent nucleic acid amplification' refers to a method of linking two types of probes through ligation and then amplifying them. Specifically, it refers to a method in which two types of probes hybridized adjacent to a target nucleic acid are linked through ligase, etc., and then nucleic acid polymerase initiates a reaction starting from the linked probe to amplify the target nucleic acid. The two types of probes can be linked by ligase only when they are completely hybridized with the target nucleic acid. The probe has a target base sequence portion that is complementary to the target nucleic acid and can hybridize, and an amplification arm that can facilitate amplification by PCR. The other probe consists of a primer sequence portion along with a target sequence portion separated by one base, and when the two types of probes are successfully hybridized to the target nucleic acid, the 3'-end and 5'-end for ligation -The ends face each other. The term 'adjacent' means that the 3'-end of one probe and the 5'-end of the other probe are sufficiently close to each other so that the ends of the two types of probes can be connected to each other. The two probes each have a complementary portion to the target nucleic acid, and when the two probes are completely hybridized or annealed to the target nucleic acid, they are linked by ligase to form one single-stranded DNA fragment. The ligated probe DNA fragment is used as a template to amplify the target nucleic acid, a labeled PCR primer is designed using the sequence in the amplification arm, and the nucleic acid is amplified by PCR using the primer. If the two probes are not hybridized and ligated to the target nucleic acid, a signal resulting from the label on the transcription product of the two probes is not ultimately generated, and therefore, amplification or detection of the target nucleic acid is not achieved.

In the present invention, the term 'target nucleic acid' may be any nucleic acid. In particular, if the target nucleic acid is a gene overexpressed in a specific disease or expressed only in a specific disease, the target nucleic acid may be a target gene for diagnosing the disease. At this time, the disease may be a disease caused by genetic mutation or chromosomal abnormality (aneuploidy), cancer, etc., but is not limited thereto. Additionally, the target nucleic acid may be derived from a pathogenic microorganism, in which case the probe set can be used to detect the pathogenic microorganism. At this time, the pathogenic microorganism refers to a microorganism that becomes an infectious agent that causes disease in the body of humans or animals, and may be a virus, bacteria, etc. The virus may be a single-stranded RNA, double-stranded RNA or DNA virus, such as Coronaviridae, Filoviridae, Orthomyxoviridae, Herpesviridae, papilomaviridae or It may be a virus belonging to the Picornaviridae family, and specifically may be an influenza virus, SARS-CoV-2 virus, or a mutant virus thereof. The bacteria may be gram-positive or gram-negative, for example, pathogenic E. coli, Salmonella, Clostridium, Streptococcus, or Staphylococcus aureus.

In the present invention, the term 'probe' refers to a base sequence containing a single-stranded oligonucleotide sequence complementary to the target nucleic acid, and is used for the synthesis of an amplification product in which nucleic acids are synthesized and extended by the isothermal nucleic acid amplification reaction of the present invention. It can act as a viewpoint. The length and sequence of the probe must be determined to begin synthesis of the extension product. The specific length and sequence of the probe can be determined depending on temperature and ionic strength conditions as well as the complexity of the target nucleic acid required.

The probe may be composed of nucleic acid, and the nucleic acid may be DNA or RNA such as gDNA, cDNA, etc. In addition, nucleotides, which are the basic structural units in the nucleic acid, may include not only natural nucleotides but also analogues in which sugar or base sites are modified. For example, it may contain phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or it may contain an intercalating agent.

In the present invention, the term 'ligation-free isothermal nucleic acid amplification', unlike the conventional ligation-dependent nucleic acid amplification method, uses complex PCR primer design and enzymes. It refers to a method that can amplify and detect trace amounts of target nucleic acid under isothermal conditions without the need for a ligation agent (ligase).

Hereinafter, the present invention will be described in detail.

1. Probe set and composition for target nucleic acid amplification

One aspect of the present invention provides a probe set for isothermal amplification of a target nucleic acid without ligation.

In addition, another aspect of the present invention provides a composition for amplifying a target nucleic acid comprising the probe set and a chain-displacement nucleic acid polymerase.

The target nucleic acid may be any nucleic acid to be amplified, and the nucleic acid may be RNA or DNA. Additionally, it may be a nucleic acid isolated from a sample subject, and may be a nucleic acid present in or isolated from whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, or cells of the sample.

The probe set of the present invention includes two types of probes, a first probe and a second probe, each capable of recognizing two different, non-overlapping regions of the target nucleic acid (see FIG. 1(a)).

The first probe and the second probe can amplify the target nucleic acid isothermally without ligation according to the principle shown in FIG. 1. Hereinafter, the configuration and operating principles of the first and second probes will be described with reference to FIG. 1.

The first probe (FW probe) includes a first base sequence complementary to the first region of the target nucleic acid, and a base sequence forming a hairpin structure connected to the 3'-end of the first base sequence.

The first base sequence is a base sequence that can bind complementary to the first region of the target nucleic acid. The first region of the target nucleic acid is any region present in the target nucleic acid. Since the first probe includes a first base sequence, it can recognize the target nucleic acid by binding complementary to the first region. The first base sequence is preferably designed to maximize hybridization to the target nucleic acid while minimizing the formation of any other structures.

In addition, a base sequence forming a hairpin structure is connected to the 3'-end of the first base sequence. The hairpin structure is also called a stem-loop structure, and is a structure formed by bonding between base pairs that occurs in a single-stranded nucleic acid. When two regions of the single-stranded nucleic acid are read in opposite directions and the base pairs are complementary to each other, a hairpin structure may be formed. The sequence of the hairpin structure may be a known hairpin structure, but is not limited thereto, and any nucleotide sequence that can form a hairpin structure because the two regions are complementary when read in opposite directions can be used without limitation.

The hairpin structure is connected to the 3'-end of the first base sequence. As described above, a hairpin structure is connected to the 3'-end of the first base sequence and serves as a starting point for nucleic acid polymerization, so the target nucleic acid can be amplified without a separate PCR primer. As shown in (a) of Figure 1, when the first probe is hybridized to the first region of the target nucleic acid through the first base sequence, the base sequence linked to the 3'-end of the first base sequence A hairpin structure is formed. As a result, the 3'-end of the first probe is folded and positioned in the same direction as the 5'-end of the first probe. In this way, the 3'-end of the hairpin structure located in the same direction as the 5'-end of the first probe can itself act as a self-primer. The above-mentioned self-primer refers to nucleic acid polymerization using the portion linked to the 5'-end of the first probe by a nucleic acid polymerase from the 3'-end of the folded hairpin structure provided by the first probe as a template. It refers to a hairpin structure that can be initiated, and the first base sequence of the 5'-part of the first probe and the sequence conjugated thereto or the adjacent sequence even if not conjugated are formed by the self-priming role of the hairpin structure. can be amplified. In this way, since the hairpin structure of the first probe acts as a self-primer, it is possible to initiate the extension reaction by nucleic acid polymerase without a separate primer (see Figures 1(a) and (b)).

Additionally, the second probe includes a second base sequence complementary to the second region of the target nucleic acid, and a base sequence encoding a hairpin structure linked to the 5'-end of the second base sequence.

The second base sequence is a base sequence that can bind complementary to the second region of the target nucleic acid. The second region is located in the 3'-end direction of the target nucleic acid. The first region and the second region of the target nucleic acid are located adjacent to each other. Because of this, the first probe and the second probe are located adjacent to each other when hybridized to the target nucleic acid, and since the two probes are not ligated, a gap (nick) exists between the two probes.

The nick refers to the absence of internucleotide linkage between two different nucleic acid fragments or a discontinuity in a nucleic acid molecule, for example, it may be the absence of a phosphodiester bond between adjacent nucleotides of one strand. there is. The description of the connection between nucleotides refers to the content described above while explaining the ligation.

For example, the first region in the target nucleic acid is located adjacent to the first region and the second region at a distance of 0 to 3, 0 to 2, or 0 to 1 bases, so that the two probes When hybridized with the target nucleic acid, a gap exists between the first probe and the second probe.

A base sequence encoding a hairpin structure is connected to the 5'-end of the second base sequence. The base sequence encoding the hairpin structure is such that the nucleic acid chain newly polymerized by nucleic acid polymerase forms a hairpin structure, and the newly polymerized hairpin structure is self-as described above when describing the first probe. It is primed and becomes the starting point for new nucleic acid polymerization reactions.

Meanwhile, the base sequence encoding the hairpin structure may be modified to reduce base overlap interactions. Through the above modification, the nucleic acid chain formed by newly polymerizing from the base sequence encoding the hairpin structure can separate without interacting with the second probe, which is a template, and form a hairpin structure more easily. In other words, through the above modification, self-priming of the nucleic acid chain newly polymerized from the second probe becomes easier. The above modification reduces the base stacking interaction of the double helix structure of at least one phosphodiester bond in the base sequence encoding the hairpin structure, thereby lowering the T _m (melting point) of the double helix nucleic acid. It may have been modified into another form. For example, the phosphodiester bond can be modified into a phosphorothioate bond. Additionally, a mismatch sequence may be introduced into the base sequence encoding the hairpin structure, or the hairpin structure may be modified with UNA (unlocked nucleic acid), etc. The portion modified to reduce the base stacking interaction may be 5 or more bases, 12 or more bases, 20 or more bases, or 25 or more bases. In a specific example of the present invention, it was confirmed that amplification of the target nucleic acid occurred efficiently when 20-25 of the base sequences encoding the hairpin structure of the second probe were phosphorothioate modified (see Figure 7).

When a target nucleic acid is present, it hybridizes to the first region of the target nucleic acid through the first base sequence of the first probe. And, by self-priming of the first probe (FW probe), nucleic acid polymerase extends the nucleic acid in the direction where the second probe (PS probe) exists (see FIG. 1(c)), and at this time, 5 of the second probe The part complementary to the '-terminus cannot be complementary to the second primer due to the base overlap interaction reduced by the modification present in the second primer, and eventually a new hairpin structure may be formed at the 3'-end ( (see Figures 1(c) and (d)).

Meanwhile, another aspect of the present invention provides a composition for amplifying a target nucleic acid including the probe set and a chain-displacement nucleic acid polymerase.

The probe set of the present invention can amplify a target nucleic acid isothermally when using chain displacement nucleic acid polymerase. The strand displacement means that the previously synthesized strand is separated from the template while a new base sequence is extended by a polymerase. By using the probe set that acts as a self-primer and chain-displacement nucleic acid polymerase, target nucleic acids can be amplified at isotherm without additional primers. The chain displacement nucleic acid polymerase includes Bst nucleic acid polymerase, Bst polymerase large fragment, Bst 2.0 WarmStart DNA polymerase, Bst 3.0 DNA polymerase, Vent(exo-) DNA polymerase, and phi29 (NEB #M0269) nucleic acid polymerase. , SD nucleic acid polymerase, exo(-) Deep vent DNA polymerase, exo(-) Pfu DNA polymerase, Bca DNA polymerase, etc., but is not limited to these, and any nucleic acid polymerase with chain displacement ability may be included without limitation. .

In particular, the chain-displacement nucleic acid polymerase may have the activity of polymerizing nucleic acid and elongating the strand even if there is a nick in the template. In this case, amplification of the target nucleic acid can occur even if the first probe and the second probe are not covalently linked by an enzymatic ligation agent, a non-enzymatic ligation agent, or a photoligation agent. Therefore, when a chain-displacement nucleic acid polymerase that can polymerize nucleic acids even if there is a gap in the template is used together with the probe set of the present invention, it can be used ligation-free or ligation-independent. Nucleic acid polymerization reactions can be carried out at isothermal temperatures. Chain displacement nucleic acid polymerases that can polymerize nucleic acids even if there are gaps in the template include Bst DNA polymerase, such as Bst DNA polymerase I, Bst polymerase large fragment, Bst 2.0 WarmStart DNA polymerase, and Bst 3.0 DNA polymerase. You can. Bst DNA polymerase is DNA polymerase I of Bacillus stearothermophilus strain, and has 5'→3' polymerase activity and double-strand-specific 5'→3' exonuclease activity, but 3' →No 5'exonuclease activity. The large fragment of Bst DNA polymerase has 5'→3' DNA polymerase activity and strong strand displacement activity, but lacks 5'→3' exonuclease activity. Bst 2.0 WarmStart DNA polymerase is a homologue synthesized in silico of a large fragment of Bst DNA polymerase, and is equipped with a reversibly bound aptamer that inhibits polymerase activity at temperatures below 45°C, making it similar to wild-type Bst DNA polymerase. It has superior amplification speed, yield, salt tolerance, and thermal stability compared to large fragments. Bst 3.0 DNA polymerase is a homolog synthesized in silico of a large fragment of Bst DNA polymerase, and nucleic acid fragments are combined to increase isothermal amplification ability and reverse transcription activity, resulting in strong performance even under high concentrations of amplification inhibitors, including dUTP. It has the characteristic of greatly increased reverse transcriptase activity compared to Bst DNA polymerase. However, it is not limited thereto, and any polymerase that has chain substitution ability and can polymerize nucleic acids in a template with a gap may be included without limitation.

In a specific embodiment of the present invention, it was discovered for the first time that Bst polymerase large fragment, Bst 2.0 WarmStart DNA polymerase, and Bst 3.0 DNA polymerase can polymerize nucleic acids using gapped nucleic acids as templates, and the Bst DNA polymerase It was confirmed that amplification of the target nucleic acid is possible at isotherm without a separate ligation reaction (see Figure 8).

The composition for target nucleic acid amplification may further include a buffer solution and deoxyribonucleotide (dNTP). The gene amplification reaction buffer is added for the amplification reaction of nucleic acids and may be a buffer containing Tris-HCl, betaine, bovine serum albumin (BSA), and Tween 20. In this case, Tween 20 Instead, it may be a buffer solution containing Triton X-100. Additionally, the buffer solution may further include magnesium chloride, magnesium sulfate, ammonium sulfate, glycerol, DSMO, TMAC, purified water, stabilizers, etc. The stabilizer may be a stabilizer commonly used in the art, such as trihalose or Methyl-a-D-Gluco-pyranoside, but is not limited thereto, and may be included without limitation as long as it stabilizes the probe set or the chain displacement nucleic acid polymerase. there is. The buffer and dNTPs must be contained in appropriate amounts to prevent nucleic acid amplification from being performed smoothly or non-specific reactions occurring. For example, it is preferable that the buffer solution is 35% by weight or less, 30% by weight or less, and 25% by weight or less based on the total weight of the composition. The dNTP is preferably 10% to 30% by weight, specifically 15% to 25% by weight, based on the total weight of the composition. If it exceeds the above range, a non-specific reaction may occur, or if it is below the above range, a nucleic acid amplification reaction may occur. This may not happen smoothly.

Referring to FIG. 1, when a target nucleic acid is present, it hybridizes to the first region of the target nucleic acid through the first base sequence of the first probe. And by self-priming of the base sequence forming the hairpin structure connected to the 3'-end of the first probe, nucleic acid polymerase rips the nucleic acid chain from the 3'-end of the hairpin structure in the direction in which the second probe exists. extend it At this time, polymerization of the nucleic acid chain may occur even if the first probe and the second probe are not ligated after hybridization to the target nucleic acid by a chain displacement nucleic acid polymerase such as Bst polymerase (Figures 1(a) and ( b) Note).

In addition, at the same time, the chain can be extended from the 3'-end of the second probe present in the nick between the two probes in the direction in which the first probe is located by the nucleic acid polymerase (Figure 1 ( c) Note). As a result, the hairpin structure (“a-loop-a'” part) of the first probe unfolds as the chain is polymerized by the chain displacement ability of nucleic acid polymerase (see Figure 1(d)). The hairpin present in the first probe, formed through the above process, is unfolded, and polymerization of nucleic acid begins again from the newly formed hairpin in the sequence complementary to the 5'-end portion of the second probe by chain displacement nucleic acid polymerase. do. As a result, the newly extended strand at the 3'-end of the second probe present in the nick is separated from the strand extended at the 3'-end of the first probe (see FIG. 1(e)). In this way, since the nucleic acid strands are separated by the chain-displacement nucleic acid polymerase, amplification of the target nucleic acid can occur isothermally without a separate heating process for denaturing the double-stranded nucleic acid.

Using the base sequence extended from the 3'-end of the second probe isolated as above as a template, the above process is repeated as the nucleic acid is polymerized by chain-displacement nucleic acid polymerase (see Figure 1(f)), and The resulting target nucleic acid can be amplified repeatedly.

In a specific example of the present invention, it was confirmed that amplification of the target nucleic acid occurred even without ligation of the two probes (see Figure 2).

In addition, in another specific example of the present invention, the intermediate product generated in the ligation-independent isothermal nucleic acid amplification reaction of the present invention was confirmed, and when the 3'-end of the second probe was blocked, amplification of the target nucleic acid did not occur. By confirming that nucleic acid polymerization also occurs at the 3'-end of the second probe present in the gap between the two probes, the principle and feasibility of the ligation-independent isothermal nucleic acid amplification reaction of the present invention were confirmed (see Figures 4 and 5 ).

In the present invention, the limitations of existing nucleic acid polymerization chain reaction (PCR) and ligation-based nucleic acid amplification reaction technologies are overcome through an isothermal nucleic acid amplification reaction based on the self-priming reaction of the probe set, such as complex design of primers, separate primers, Target nucleic acids can be easily and conveniently amplified using only two types of probes without an expensive temperature control device.

In a specific embodiment of the present invention, a first probe containing a base sequence forming a hairpin structure and a second probe containing a base sequence encoding a hairpin structure modified to reduce base overlapping interactions are introduced into the target nucleic acid. Target nucleic acid can be detected without additional primers under isothermal conditions through self-polymerization of the hairpin probe induced by hybridization with and amplification of target nucleic acid due to non-hybridization of PS-DNA/DNA structure by phosphorothioate DNA. It was confirmed that this was possible (see Figure 2).

2. Kit for detecting target nucleic acids

Another aspect of the present invention is a composition for detecting a target nucleic acid, comprising a composition for amplifying a target nucleic acid, a gRNA that binds to at least a portion of the target nucleic acid, a Cas12 enzyme, and a single-stranded nucleic acid probe labeled at both ends with a fluorescent dye and a quencher. Kits are provided.

The description of the target nucleic acid composition and target nucleic acid refers to the above description and is not repeated.

The gRNA is an RNA that specifically recognizes a target nucleic acid, and is expressed through transcription of a recombinant nucleic acid construct delivered into the cell to recognize the target gene, form a complex with the Cas12 protein, and bring the Cas12 protein to the target nucleic acid. . The gRNA includes a spacer capable of recognizing a target nucleic acid; And it may include a guide RNA scaffold consisting of a non-variable sequence unrelated to the target. The spacer refers to a sequence that allows gRNA to recognize a target nucleic acid, and may include part or all of the PAMs sequence near the target nucleic acid position. Preferably, sequences adjacent to the PAMs (Protospacer Adjacent Motif sequences) can be used. Additionally, the spacer may be modified appropriately depending on the type of target cell. The gRNA recognizes adjacent sequences from the PAMs region and induces cleavage, with very high specificity.

The gRNA may include a base sequence complementary to at least a portion of the target nucleic acid. Additionally, it may include sequences complementary to both part of the first region and part of the second region of the target nucleic acid. When designing a gRNA as described above, the target nucleic acid sequence amplified by the probe set can be selectively recognized, and a sequence identical to the target nucleic acid, such as the target nucleic acid, can be recognized to induce cleavage of a single DNA strand. can do.

The Cas12 enzyme is one of the type 5 CRISPR effector proteins. When the target DNA binds, it cleaves both ends of the DNA and has a trans-cleavage activity that randomly decomposes the surrounding single DNA strand, that is, ssDNA that does not hybridize to gRNA. Have. Using this activity, a fluorescent dye and quencher linked to a single DNA strand can be added around Cas12 that reacts with the target nucleic acid, thereby inducing DNA decomposition and fluorescence emission. The Cas12 enzyme may be Cas12a (Cpf1), Cas12b (C2c1), Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i or Cas12j, and specifically may be Cas12a (Cpf1). For example, Cas12a may be LbCas12a, AsCas12a, EeCas12a, etc. However, it is not limited to this, and any CRISPR effector protein that recognizes a double-stranded target nucleic acid by binding to gRNA and has trans-cutting activity can be used without limitation.

In the present invention, Cas12 enzyme refers to Cas12 protein or a vector expressing Cas12 protein. Additionally, the Cas12 protein or the vector expressing the Cas12 protein may be in a form that allows the Cas protein to function in the nucleus. Cas12 protein refers to an essential protein element in the CRISPR/Cas12 system, and can act as an active endonuclease or nickase when forming a complex with gRNA. Information on the Cas12 gene and protein can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

The kit for detecting target nucleic acids may further include excipients that stabilize gRNA and Cas12 protein. The excipient may be a buffer, NEBuffer, NaCl, tris-HCl, MgCl2, albumin, salt, acid, or base, but is not limited thereto, and any excipient commonly known to stabilize the CRISPR/Cas12a composition may be used.

The single-stranded nucleic acid probe includes a fluorescent dye and a quenching agent. The fluorescent dye and quenching agent may each be independently located at the 5'-end, 3'-end, and center of the nucleic acid probe. The single nucleic acid probe is a FRET (fluorescence resonance energy transfer) type in which the energy of the fluorescent dye is transferred to a quencher (fluorescence suppressor) before cleavage, suppressing fluorescence signal emission, and after cleavage of the probe, no fluorescence signal transfer occurs. It is a signal probe. Additionally, the base sequence included in the single-stranded probe has a base sequence that is non-complementary to the target nucleic acid and the probe. The length of the nucleic acid probe may be 3-nt to 300nt, 5nt to 100nt, 6nt to 50nt, or 8nt to 20nt.

When the single-stranded nucleic acid probe is added to the isothermal amplification reaction product, the Cas12/gRNA binds to the target nucleic acid to create a Cas12/gRNA/target nucleic acid triple complex, and the single-stranded DNA probe is cleaved by the complex, causing fluorescence of the fluorescent dye. can be detected. Therefore, if the same sequence as the target nucleic acid is present in the isothermal amplification reaction product, a fluorescence signal is confirmed, and if a non-specific nucleic acid is present, the triple complex of Cas12/gRNA/target nucleic acid is not generated and no fluorescence signal is confirmed (Figure 1 ( g) see).

In a specific embodiment of the present invention, as a result of performing the ligation-independent isothermal nucleic acid amplification reaction of the present invention using analysis samples containing target nucleic acids at various concentrations (1 fM ~ 100 pM), target nucleic acid detection of the technology of the present invention The limit of detection (LOD) was confirmed to be 49.2fM, confirming that the nucleic acid amplification technology of the present invention has excellent sensitivity (see Figure 12).

In another specific embodiment of the present invention, through the ligation-independent isothermal nucleic acid amplification technology of the present invention, not only can any non-specific nucleic acid base sequence be distinguished, but also 1 to 3 mismatched bases or 1 to 3 mismatched bases. It was confirmed that even cases where there is a gap between probes could be successfully distinguished, and through this, it was confirmed that the target nucleic acid amplification and detection technology of the present invention has excellent selectivity or specificity for the target nucleic acid (see Figure 11).

3. Amplification method and detection method of target nucleic acid

Another aspect of the present invention provides a method of amplifying a target nucleic acid at isotherm using the composition for amplifying the target nucleic acid.

The target nucleic acid amplification method of the present invention may include the step of mixing the target nucleic acid with the composition for amplifying the target nucleic acid and performing a nucleic acid polymerization reaction at isothermal.

When the target nucleic acid is mixed with the target nucleic acid amplification composition, the target nucleic acid, the probe set of the present invention, and the chain displacement nucleic acid polymerase exist in the same reaction space. As described above, when the first probe and the second probe are hybridized to the first region and the second region of the target nucleic acid, respectively, in the same reaction space, from the hairpin structure present at the 3'-end of the first probe as described in FIG. 1 Polymerization of the nucleic acid chain is initiated. At this time, the reaction must be performed so that nucleic acid polymerization sufficiently occurs at a temperature that does not denature the chain-displacement nucleic acid polymerase or does not substantially inhibit the activity of the chain-displacement nucleic acid polymerase. In addition, the temperature at which the target nucleic acid is amplified is such that the probe set can hybridize with the target nucleic acid and the newly synthesized sequence can be self-primed by modification in which the base overlap interaction of the second probe is reduced. The temperature should be around this level. For example, it may be 40-80°C, 50-70°C, or 55-65°C.

In addition, as shown in Figure 1, the amplification method of the target nucleic acid involves self-priming by the probe set and separation of the nucleic acid by chain-displacement nucleic acid polymerase, so a separate heating process is required to denature the double-stranded nucleic acid. Otherwise, it may be performed under isothermal conditions where any temperature selected from the above temperature range is maintained constant. However, if the target nucleic acid is a nucleic acid with a double helix structure such as DNA, an additional preheating process to denature the double helix into a single strand may be necessary before reacting the probe set with the target nucleic acid. However, even in this case, after adding the probe set, the target nucleic acid can be amplified under isothermal conditions in which any temperature condition selected from the above temperature range is maintained constant.

The isothermal amplification method of the target nucleic acid takes about 20 minutes or more, 30 minutes or more, 40 minutes or more, or about 1 hour from DNA extraction of the sample to complete amplification, and if separation or extraction of nucleic acids from the sample is performed, about 20 to 20 minutes. It has the advantage of being able to perform the amplification reaction very quickly, taking about 40 minutes.

In the method of amplifying the target nucleic acid, the concentration of the chain displacement nucleic acid polymerase must be sufficient to polymerize the target nucleic acid. For example, the concentration of the nucleic acid polymerase may be 5U or more, 10U or more, 20U or more, or 40U or more, but is not limited thereto and can be appropriately selected depending on the concentration of the target nucleic acid present.

In the method of amplifying the target nucleic acid, the probe set concentration must be sufficient to recognize the target nucleic acid. For example, the concentration of the probe set may be 1 nM or more, 10 nM or more, or 50 nM or more, but is not limited thereto and can be appropriately selected depending on the concentration of the target nucleic acid present.

If the method for amplifying the target nucleic acid is carried out at a temperature, concentration of the probe, and concentration of the chain displacement nucleic acid polymerase within the above range, the target nucleic acid can be efficiently amplified, but is not limited to the above range. You can select it by adjusting it appropriately.

In a specific example of the present invention, through optimization experiments of the ligation-independent isothermal nucleic acid amplification reaction of the present invention, experiments were performed by selecting a probe concentration of 10 nM and 20 U of Bst nucleic acid polymerase at a temperature of 60°C (Figure 7 to 10).

The ligation-independent isothermal nucleic acid amplification technology of the present invention is effective in continuously amplifying target nucleic acids, especially RNA, without a reverse transcription step, and uses only two types of probes and a chain-displacement nucleic acid polymerase without complex design of multiple primers. Thus, the target nucleic acid can be amplified.

In addition, target nucleic acids can be amplified multiple times using the target nucleic acid amplification composition that recognizes different target nucleic acids.

In addition, another aspect of the present invention includes amplifying a target nucleic acid using the composition for amplifying a target nucleic acid; and generating a fluorescent signal using a single-stranded nucleic acid probe containing gRNA, Cas12, a fluorescent dye, and a quencher that binds to at least a portion of the target nucleic acid.

The step of generating the fluorescence signal may be performed independently or together with the step of amplifying the target nucleic acid.

When the target nucleic acid is present and the target nucleic acid is amplified, the gRNA/Cas12/target nucleic acid triple complex that specifically recognizes the target nucleic acid is activated and can cleave the single-stranded nucleic acid probe containing the fluorescent dye and quencher. Before the single-stranded nucleic acid probe is cleaved, the fluorescence energy of the fluorescent dye is transferred to a quencher (fluorescence suppressor), thereby suppressing the emission of the fluorescent signal. However, after the probe is cleaved by the activated triple complex, no fluorescence signal transfer occurs. A fluorescence signal is generated by the released fluorescent dye, allowing the presence or absence of the target nucleic acid to be confirmed with the naked eye (see Figure 1(g)). Therefore, when the target nucleic acid is present, the fluorescent dye is separated from the quencher, and a fluorescent signal is generated from this, the process may include determining that the target nucleic acid is present. The generation of the fluorescent signal may result in a wavelength shift (or color shift) of the detectable signal due to the release of the fluorescent dye, and may be expressed as a decrease in the signal of a specific wavelength and an increase in the signal of another wavelength.

In addition, since multiple amplification of different target nucleic acids is possible as described above, multiple detection of target nucleic acids is possible using gRNA that recognizes different target nucleic acids.

In a specific embodiment of the present invention, different target nucleic acids are selectively amplified from nucleic acid samples containing non-specific nucleic acids, total RNA extracted from actual cells, total RNA extracted from animal tissues, and RNA from exosomes extracted from animal urine. Thus, it was confirmed that multiple target nucleic acids can be effectively detected not only in nucleic acid samples but also in liquid biopsies (see FIGS. 12, 14, and 15).

The ligation-independent isothermal nucleic acid amplification and target nucleic acid detection technology based on the present invention have high sensitivity and enable simultaneous detection of multiple target nucleic acids even in complex body fluid samples. These results can be useful for body fluid biopsies that do not require invasive procedures, and can also be used for molecular-level diagnosis of diseases. In addition, the present invention has the advantage of being able to analyze multiple target nucleic acids through ligation-independent isothermal nucleic acid amplification, so it can be used for disease prevention and early diagnosis through analysis of various target nucleic acids.

Hereinafter, the present invention will be described in detail.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

[1-1] Preparation of samples

All oligonucleotides were purchased from IDT (Integrated DNA Technology) (Coralville, IA, USA), and the sequences used in the experiment are shown in Table 1. Bst DNA polymerase large fragment, Bst 2.0 WarmStart DNA polymerase, Bst 3.0 DNA polymerase, Vent(exo -) DNA polymerase, 10X Cutsmart buffer and dNTPs were from New England Biolabs Inc. (NEB, Beverly, MA, USA). Purchased. The miRNeasy Mini Kit (217004) was purchased from Qiagen (Hilden, Germany). Gel-Red Nucleic Acid Stain (41003) was purchased from Biotium (Fremont, CA, USA). One Step TB Green PrimeScript RT-PCR kit was purchased from Takara Korea Biomedical Inc. (Seoul, Korea).

In Table 1 below, the underlined part indicates the part that forms the hairpin structure in the FW probe sequence, and the asterisk (*) indicates the part of the sequence that encodes the hairpin structure in which the phosphorothioate modification is introduced in the PS probe sequence.

sequence number NAME Sequence(5'→3') Size(bp) One ERBB2 RNA GAG AGG GGA AGC GGC CCU AAG GGA GUG UCU AAG AAC AAA AGC GAC CCA U 49 2 ERBB2 FW probe /5Phos/A CTC CCT TAG GGC CGC TTC CCC TCT TAG AAC AAT TAA TTG TTC TA 45 3 ERBB2 FW - one base mismatch /5Phos/T CTC CCT TAG GGC CGC TTC CCC TCT TAG AAC AAT TAA TTG TTC TA 45 4 ERBB2 FW - two bases mismatch /5Phos/T GTC CCT TAG GGC CGC TTC CCC TCT TAG AAC AAT TAA TTG TTC TA 45 5 ERBB2 FW - three bases mismatch /5Phos/T GAC CCT TAG GGC CGC TTC CCC TCT TAG AAC AAT TAA TTG TTC TA 45 6 ERBB2 FW - one space /5Phos/ CTC CCT TAG GGC CGC TTC CCC TCT TAG AAC AAT TAA TTG TTC TA 44 7 ERBB2 FW - two space /5Phos/ TCC CTT AGG GCC GCT TCC CCT CT T AGA ACA ATT AAT TGT TCT A 43 8 ERBB2 FW - three space /5Phos/ CCC TTA GGG CCG CTT CCC CTC T TA GAA CAA TTA ATT GTT CTA 42 9 ERBB2 PS probe(PS12) A*A*G* A*A*T* A*T*T* C*T*T* ATG GGT CGC TTT TGT TCT TAG AC 35 10 ERBB2 PS probe(PS20) A*A*G* A*A*T* T*C*T* T*A*A* G*A*A* T*T*C* T*T*ATG GGT CGC TTT TGT TCT TAG AC 43 11 ERBB2 PS probe(PS25) T*A*A* T*G*A* A*T*C* A*T*T* C*A*A* T*G*A* T*T*C* A*T*T* A* ATG GGT CGC TTT TGT TCT TAG AC 48 12 3'Block PS probe A*A*G* A*A*T* T*C*T* T*A*A* G*A*A* T*T*C* T*T*A TGG GTC GCT TTT GTT CTT AGA C/ 3Bio/ 43 13 ERBB2 PS probe(w/o PS) AAG AAT TCT TAA GAA TTC TTA TGG GTC GCT TTT GTT CTT AGA C 43 14 ERBB2 Cas12a crRNA /AltR1/UA AUU UCU ACU AAG UGU AGA UUU CUU AGA CAC UCC CUU AGG G/AltR2/ 42 15 Rep-Cas12a-FAM /56-FAM/TT ATT /3IABkFQ/ 5 16 GRBB7 RNA CUU UCU GCA GCU GCG GGG UUC AGG ACG GAA GCU UUG GAA ACG CUU UUU CU 50 17 GRBB7 FW probe /5phos/C GTC CTG AAC CCC GCA GCT GCA G AG AAC AAT TAA TTG TTC TA 42 18 GRBB7 PS probe(PS20) A*A*G* A*A*T* T*C*T* T*A*A* G*A*A* T*T*C* T*T*A GAA AAA GCG TTT CCA AAG CTT C 43 19 GRBB7 Cas12a crRNA /AlTR1/UA AUU UCU ACU AAG UGU AGA UCA AAG CUU CCG UCC UGA ACC C/AlTR2/ 42

[1-2] Cas12a protein purification

Cas12a protein was expressed using a pMAL-based vector containing N-terminal maltose binding protein (MBP), 6x His-Tag, and TEV protease cleavage site. Purified Cas12a protein was stored in storage/reaction buffer consisting of 20mM Tris-HCl pH 7.5, 100mM KCl, and 5% glycerol. Cas12a/gRNA complex was formed by reacting 100nM Cas12a protein and 150nM gRNA in 1x NEBuffer 2.1 at 25°C for 20 minutes.

[Example 2]

Confirmation of target nucleic acid detection through ligation-independent isothermal nucleic acid amplification method

It was confirmed whether specific detection of the target nucleic acid was possible through the ligation-independent isothermal gene amplification reaction of the present invention.

Specifically, ligation-independent nucleic acid amplification in a final volume of 20 μL containing reaction buffer (50mM Tris-HCl, 12mM MgCl ₂ , 1mM ATP, 10mM DTT, pH 7.6), 20U of Bst DNA polymerase large fragment, and 500nM dNTPs. The reaction was carried out. 10 nM first probe (FW probe), 10 nM second probe (PS probe) for target nucleic acid, and target RNA were reacted at 60°C for 50 minutes. At this time, to confirm that ligation-independent isothermal nucleic acid amplification occurs under different component conditions, FW and PS probes were first mixed with or without target RNA, and incubated at 37°C for 20 minutes in the presence or absence of SplintR ligase. It was allowed to react for a while.

After the reaction, 50 nM LbCas12a/crRNA complex and 500 nM fluorophore-quencher conjugate (5'-FAM-TTATT-3IABkFQ-3') previously formed in Example 1 were added to 3 μL of the reaction product, and incubated at 37°C in CFX Opus 96 RT. -The fluorescence signal was monitored in real time at 1-minute intervals for 40 minutes using a PCR system (Bio-Rad, CA, USA).

In addition, electrophoresis was performed on a mixture of 20 μL of the target nucleic acid reactant and 6x agarose gel loading buffer using a 2% agarose gel containing Gel-Red Nucleic Acid Stain. Nucleic acid amplification products were visualized using the Geldoc Go Imaging system (Bio-Rad, CA, USA).

As a result, as shown in Figure 2(a), it was confirmed that an isothermal gene amplification reaction occurred regardless of the presence or absence of ligase and that the fluorescence signal was amplified in real time. When the FW and PS probes were mixed with the target RNA without Bst DNA polymerase, no signal was detected (1 and 2 in Figure 2(a)), and when the Bst DNA polymerase was mixed with the FW and PS probes and the target RNA, the ligase The fluorescence signal increased regardless of the presence or absence of the enzyme (3 in Figure 1(a)). This indicates that repetitive DNA expansion containing the target RNA sequence can occur without ligation, as shown in Figure 1. When the ligation and polymerization steps were sequentially reacted with the probe and target RNA, the fluorescence signal was measured to gradually increase (4 in Figure 2(a)). This suggests that continuous DNA extension through PS-THSP is possible after the two probes are hybridized to the target RNA regardless of the ligation reaction. Conversely, no increase in fluorescence was observed in the absence of target RNA (5-8 in Figure 2(a)). This result suggests that nucleic acid polymerization by the two types of probes occurs after hybridization with the target RNA.

In addition, as shown in Figure 2(b), it was clearly confirmed again through electrophoresis that the target nucleic acid was amplified even though ligase was not used. Bands representing long extensions of the DNA structure were observed only when the probe and target RNA were reacted with Bst DNA polymerase (3 and 4 in Figure 2(b)), which is consistent with the results of the fluorescence signal measurement. In addition, as a result of performing ligation-independent nucleic acid amplification in the same manner as above under the conditions of the presence or absence of the target and the presence or absence of various probes, the fluorescence signal increased only in the presence of the target RNA and the FW and PS probes, as shown in Figure 3. It was confirmed that the ligation-independent isothermal nucleic acid amplification method of the invention can be usefully used for RNA detection.

[Example 3]

Verification of feasibility of ligation-independent isothermal nucleic acid amplification reaction

[3-1] Identification of intermediate products according to the presence or absence of target nucleic acid and nucleic acid polymerase

To confirm the intermediate product of the ligation-independent isothermal nucleic acid amplification method of the present invention, nucleic acid polymerization reaction was performed at low temperature.

Ligation and polymerization reactions were performed at low temperature to identify the reaction intermediate. First, the FW and PS probes were mixed with or without target RNA, and the ligation reaction was performed for 20 minutes in the presence or absence of ligase at 37 °C. Next, the polymerization reaction was performed at 45°C for 40 minutes with or without Bst DNA polymerase. Although DNA polymerization can occur at a temperature of 45°C, the PS-THSP reaction cannot proceed because the temperature is not high enough to destabilize or dissociate the polymer of the extended PS probe. Afterwards, electrophoresis was performed in the same manner as in Example 2.

As a result, as shown in Figure 4, when the probe was mixed with the target RNA, a hybridized structure was observed (1 in Figure 4). In the absence of target RNA, only a band for the probe was observed (5 in Figure 4). Afterwards, when Bst DNA polymerase was added, the extended DNA structure could be confirmed regardless of the presence or absence of ligation (3 and 4 in Figure 4), and if the target nucleic acid was not present, the amplification product was obtained regardless of the presence or absence of polymerase and ligase. It was confirmed that no intermediates were produced (7 and 8 in FIG. 4).

[4-2] Check whether nucleic acid is polymerized in the nick located between the two probes

In order to confirm whether nucleic acid polymerization occurs at the nick located at the 3'-end of the second probe (PS probe) in the ligation-independent isothermal nucleic acid amplification method of the present invention, a second probe whose 3'-end is blocked with biotin is used. After preparing the microbe, the nucleic acid was amplified in the same manner as in Example 2, and the fluorescence signal was measured in real time.

As a result, as shown in Figure 5, when the 3'-end of the second probe was blocked with biotin, the fluorescence signal was significantly reduced compared to when an unblocked probe was used. From these results, it was found that the ligation-independent isothermal nucleic acid amplification method of the present invention simultaneously occurs nucleic acid polymerization not only in the folded portion due to self-priming, but also in the gap located at the 3'-end of the second probe. Through this, it was confirmed that the ligation-independent nucleic acid isothermal amplification reaction of the present invention is feasible and can therefore be used to amplify and detect target nucleic acids.

[Example 4]

Optimization of ligation-independent isothermal nucleic acid amplification reactions

In order to optimize the ligation-independent nucleic acid amplification reaction of the present invention, the length of the sequence (hereinafter PS sequence) into which the phosphorothioate modification is introduced in the second probe (PS probe), the concentration of the probe, and the type of nucleic acid polymerase are adjusted. The amplification efficiency was compared.

Since the PS-THSP reaction is greatly affected by the length of the PS sequence, in order to compare the efficiency of the nucleic acid amplification reaction of this outbreak according to the length of the base sequence into which the PS modification is introduced, first, PS sequences of different lengths (12, 20 and 25 bases) and a phosphodiester sequence (20 bases) were prepared. An isothermal nucleic acid amplification reaction was performed in the same manner as in Example 2 using the PS probe prepared above, and the fluorescence signal was measured.

As a result, as shown in Figure 6, the highest fluorescence signal was observed when using a PS probe containing 20 PS sequences.

Based on the above results, a PS probe was designed to have a base sequence into which a 20-base phosphorothioate modification was introduced, and then ligation-independent nucleic acid isothermal amplification reaction (PS-THSP) was performed at various temperatures ranging from 50 to 65°C. It was carried out under these conditions. Below 50°C, the amplification efficiency of the PS-THSP reaction was greatly reduced because the extended DNA strand hybridized with the PS sequence rather than self-folding. It was confirmed that the PS-THSP reaction occurred above 55°C, and the amplification reaction of the present invention The optimal temperature was selected as 60°C (see Figure 7).

Additionally, to compare the reaction efficiency for different DNA polymerases, four types of enzymes ( Bst DNA polymerase large fragment, Bst 2.0 WarmStart DNA polymerase, Bst 3.0 DNA polymerase, and Vent(exo-) DNA polymerase) were used. An isothermal nucleic acid amplification reaction was performed in the same manner as in Example 2, and the fluorescence signal was measured.

The large fragment of Bst DNA polymerase showed a rapid increase in fluorescence signal during the CRISPR/Cas12-cleavage reaction, and no increase in signal was observed for Vent(exo-) DNA polymerase because the reaction temperature (60°C) was not suitable for the enzyme ( Figure 8). Likewise, the same optimization experiment as above was performed with various polymerase and probe concentrations (FIGS. 9 and 10), and based on the results, 20U Bst DNA polymerase large fragment and 10nM probe were used.

[Example 5]

Confirmation of sensitivity of ligation-independent isothermal gene amplification reaction

[5-1] Check sensitivity according to probe design

It was confirmed whether the ligation-independent isothermal amplification-based target nucleic acid amplification method of the present invention can selectively amplify target nucleic acids by checking whether gene amplification occurred depending on the distance between the two probes and the number of mismatched sequences of the probes.

Specifically, the distance between the two types of probes hybridized to the target nucleic acid was designed to differ by 0 to 3 bases, the nucleic acid was amplified using the same experimental method as in Example 2, and the fluorescence signal was measured in real time. In addition, the number of mismatch sequences occurring between the probe and target RNA was designed to be 0 to 3, and the fluorescence signal was measured in the same manner.

As a result, it was confirmed that the highest fluorescence signal was shown when the two probes and the target nucleic acid were perfectly complementary, and that a weak fluorescence signal was shown when even one base gap existed (see Figure 11(a)). In addition, it was confirmed that when the probe and target nucleic acid had even one mismatched base, a weak fluorescence signal was displayed (see Figure 11(b)). From this, it was found that the target nucleic acid detection method based on the ligation-independent isothermal gene amplification method of the present invention works when two types of probes perfectly match the target nucleic acid, and can selectively detect the target nucleic acid.

[5-2] Confirmation of sensitivity to target nucleic acid

In order to confirm the sensitivity of the ligation-independent isothermal nucleic acid amplification-based target nucleic acid detection method of the present invention, the concentration of the target nucleic acid (1 fM to 100 pM) was varied and the fluorescence signal was measured in real time using the same experimental method as in Example 2 above. Measured.

As a result, as shown in Figure 12, it was confirmed that as the concentration of the target nucleic acid increased, the speed at which the fluorescence signal appeared was faster and its intensity was also the strongest. Furthermore, it was confirmed that the concentration of the target nucleic acid can be quantified in proportion to the concentration of the target nucleic acid (y = 0.6112x + 10.3239, R ² = 0.9622). The limit of detection (LOD) calculated through this was 49.2fM according to the standard deviation, confirming that even trace amounts of target nucleic acid can be detected through the detection method of the present invention.

In addition, three types of RNA (GAPDH, GRB7, and PPP1R1B) and non-target ssDNA were prepared as non-target nucleic acid samples to check whether target nucleic acid could be selectively detected.

As a result, as shown in Figure 12(d), almost no fluorescence signal was observed for non-target nucleic acids, and it was confirmed that a fluorescence signal was generated only for the target nucleic acid, ERBB2 RNA. From these experimental results, it was found that the ligation-independent isothermal nucleic acid amplification method of the present invention specifically amplifies only the target nucleic acid.

[Example 6]

Confirmation of simultaneous detection of two types of target nucleic acids using ligation-independent isothermal gene amplification reaction

It was confirmed that simultaneous detection of two types of target nucleic acids was possible using the ligation-independent isothermal nucleic acid amplification method of the present invention.

Specifically, different target RNA samples (control, ERBB2, GRB7, ERBB2, GRB7) were prepared, and first and second probes complementary to two types of genes (ERRB2 RNA, GRB7 RNA) were prepared, respectively. A reaction buffer containing two pairs of probes, Bst DNA polymerase, and dNTPs was mixed with the RNA sample and reacted at 60°C for 50 minutes. The final reaction was mixed with Cas12a/gRNA that recognizes the ERBB2 and GRB7 genes, respectively, and the fluorescence signal was measured in real time at 37°C.

As a result, as shown in Figure 13, when Cas12a/gRNA for ERBB2 and the reaction were mixed, the fluorescence signal selectively increased in the sample containing the ERBB2 sequence (ERBB2 and mixture). Likewise, when the reaction was mixed with Cas12a/gRNA against GRB7, the fluorescence signal increased selectively in samples containing the GRB7 sequence (GRB7 and mixture). These results show that the target nucleic acid detection method of the present invention can effectively detect target nucleic acids in a single tube, and furthermore, can selectively detect target nucleic acids simultaneously due to the specificity of the probe.

[Example 7]

Confirmation of multiple detection of target nucleic acids through actual cell experiments

It was confirmed whether two types of target nucleic acids can be simultaneously detected even in actual cells using an analysis method based on the ligation-independent isothermal nucleic acid amplification method of the present invention.

Specifically, total RNA samples extracted from HER2-positive HCC1954 cells and HER2-negative HCC1143 cells were prepared, respectively. HCC1954 and HCC1143 cells were cultured in RPMI-1640 medium (11835-030, Thermo Scientific, MA, USA) containing 10% fetal bovine serum (26140-079, Thermo Scientific) and 5% antibiotics (15240-062, Thermo Scientific). Cultured at 37°C. Total RNA was extracted using the miRNeasy Mini Kit (Qiagen), and the quality and quantity of the extracted RNA were confirmed using the Nanodrop instrument (Thermo Scientific).

RNA extracted from the cells was amplified and quantitatively analyzed using the One step TB Green PrimeScript RT-PCR kit according to the manufacturer's protocol. qRT -PCR was performed at 42°C for 5 minutes and reverse transcription at 95°C for 10 seconds, maintaining a total volume of 27 μL. Next, PCR was performed with 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C. Expression levels of target genes were normalized by the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Afterwards, it was confirmed whether simultaneous detection of the target nucleic acids ERBB2 and GRB7 was possible using the same method as in Example 6 (FIG. 14a).

As a result, as shown in Figure 14, the fluorescence response of the HCC1954 sample was faster than that of HCC1143, meaning that ERBB2 and GRB7 genes were overexpressed in HCC1954 cells. The results of analyzing the same cellular RNA sample using quantitative RT-PCR (qRT -PCR) were correlated with the ligation-independent nucleic acid amplification results (see Figure 14(c)). Through this, it was confirmed that simultaneous detection and analysis of target nucleic acids is possible even in real cells.

[Example 8]

Multiple detection and analysis of target nucleic acids through animal tissue samples and body fluid samples

It was confirmed that the ligation-independent isothermal nucleic acid amplification method of the present invention can detect multiple target nucleic acids in actual animal tissue and body fluid samples.

All animal experiments were performed with approval from the Yonsei Laboratory Animal Center Institutional Animal Care and Use Committee (IACUC 2017-0329). Four-week-old female BALB/c nude mice (N = 10, average body weight 20 ± 2 g) were purchased from Central Lab Animal Inc. (Seoul, Korea). Mice were acclimatized to temperature (22 ± 2°C), humidity (approximately 60%), and lighting (12-hour light:12-hour dark cycle) for 1 week. A special diet (Pico Lab 5053, LabDiet, CA, USA) and sterilized water were provided ad libitum throughout the experiment. Breast cancer cells, HCC1954 cells (6 × 10 ⁶ /60 μL serum-free medium) were injected into the mammary skin of female BALB/c nude mice using a 29G insulin syringe per mouse. The mouse animal model was observed until the tumor volume expanded to 1000 mm ³ or until ulceration occurred. The implanted tumor size was evaluated three times per week, and tumor size was calculated using the formula (4/3) × π × (short axis/2) ² × (long axis/2) mm ³ . If the tumor size was larger than 2 cm diagonally, the mouse was euthanized with CO ₂ . After observing tumor growth, the liver, lung, kidney, and spleen of mice with tumors and normal control mice were excised for RNA analysis. The excised tumor tissues and organs were homogenized into small pieces, and then total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. The extracted total RNA was compared for ERBB2 and GRB7 gene expression levels between each organ and tumor tissue of normal and tumor-bearing mice using a ligation-independent gene amplification method in the same manner as in Example 6.

Additionally, urine samples voluntarily urinated by the breast cancer mouse animal model were collected using a 1.5 mL tube. The urine sample was centrifuged at 300xg for 10 minutes to remove cells, and then the separated supernatant was centrifuged again at 16,000xg for 30 minutes to remove remaining cell debris and stored at 4°C. Exosomes from urine samples were isolated using the Exo-spin Exosome Purification system (Cell Guidance Systems, St. Louis, MO) according to the manufacturer's instructions, and exosome RNA was extracted using the miRNeasy Mini Kit (Qiagen). . ERBB2 and GRB7 gene expression levels were measured using a ligation-independent gene amplification method in the same manner as the cell and tissue experiment.

As a result, as shown in Figure 15. For the tumor genes ERBB2 and GRB7 genes (ERRB2 RNA, GRB7 RNA), strong fluorescent signals were measured faster in tumor tissue than in normal tissue, indicating that they were overexpressed in tumor tissue. This was consistent with the qRT-PCR results (FIG. 16). In addition, it was confirmed that both types of target genes were overexpressed in exosomes extracted from the urine of tumor-bearing mice compared to the urine of normal mice (see Figure 15(d)). The ligation-independent isothermal nucleic acid amplification method of the present invention can specifically detect multiple target nucleic acids in mouse tissues and urine samples of tumor-bearing mice, so the ligation-independent nucleic acid amplification and detection method of the present invention can be used for tissue diagnosis and liquid biopsy. It was confirmed that it can be used.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Ligation-free isothermal nucleic acid amplification for detection of target nucleic acid <130> 2022-DPA-4619 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> ERBB2 RNA <400> 1 gagaggggaa gcggcccuaa gggagugucu aagaacaaaa gcgacccau 49 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe <220> <221> stem_loop <222> (26)..(45) <400> 2 actcccttag ggccgcttcc cctcttagaa caattaattg ttcta 45 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - one base mismatch <220> <221> stem_loop <222> (26)..(45) <223> ERBB2 FW probe - one base mismatch <400> 3 tctcccttag ggccgcttcc cctcttagaa caattaattg ttcta 45 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - two bases mismatch <220> <221> stem_loop <222> (26)..(45) <400> 4 tctcccttag ggccgcttcc cctcttagaa caattaattg ttcta 45 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - three bases mismatch <220> <221> stem_loop <222> (26)..(45) <400> 5 tgacccttag ggccgcttcc cctcttagaa caattaattg ttcta 45 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - one space <220> <221> stem_loop <222> (25)..(44) <400> 6 ctcccttagg gccgcttccc ctcttagaac aattaattgt tcta 44 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - two space <220> <221> stem_loop <222> (24)..(43) <400> 7 tcccttaggg ccgcttcccc tcttagaaca attaattgtt cta 43 <210> 8 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 FW probe - three space <220> <221> stem_loop <222> (23)..(42) <400> 8 cccttagggc cgcttcccct cttagaacaa ttaattgttc ta 42 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 PS probe(PS12) <220> <221> variations <222> (1)..(12) <223> phosphorothioate modification <400> 9 aagaatattc ttatgggtcg cttttgttct tagac 35 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 PS probe(PS20) <220> <221> variations <222> (1)..(20) <223> phosphorothioate modification <400> 10 aagaattctt aagaattctt atgggtcgct tttgttctta gac 43 <210> 11 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 PS probe(PS25) <220> <221> variations <222> (1)..(25) <223> phosphorothioate modification <400> 11 taatgaatca ttcaatgatt cattaatggg tcgcttttgt tcttagac 48 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> 3'-Block PS probe <220> <221> variations <222> (1)..(20) <223> phosphorothioate modification <400> 12 aagaattctt aagaattctt atgggtcgct tttgttctta gac 43 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 PS probe (w/o PS) <400> 13 aagaattctt aagaattctt atgggtcgct tttgttctta gac 43 <210> 14 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> ERBB2 Cas12a crRNA <400> 14 uaauuucuac uaaguguaga uuucuuagac acucccuuag gg 42 <210> 15 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Rep-Cas12a-FAM <400> 15 ttatt 5 <210> 16 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> GRBB7 RNA <400> 16 cuuucugcag cugcgggguu caggacggaa gcuuuggaaa cgcuuuuucu 50 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> GRBB7 FW probe <220> <221> stem_loop <222> (24)..(42) <400> 17 cgtcctgaac cccgcagctg cagagaacaa ttaattgttc ta 42 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> GRBB7 PS probe(PS20) <220> <221> variations <222> (1)..(20) <223> phosphorothioate modification <400> 18 aagaattctt aagaattctt agaaaaagcg tttccaaagc ttc 43 <210> 19 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> GRBB7 Cas12a crRNA <400> 19 uaauuucuac uaaguguaga ucaaagcuuc cguccugaac cc 42

Claims (12)

A first probe comprising a first base sequence complementary to the first region of the target nucleic acid and a base sequence forming a hairpin structure linked to the 3'-end of the first base sequence; and
A second probe comprising a second base sequence complementary to the second region of the target nucleic acid and a base sequence encoding a hairpin structure linked to the 5'-end of the second base sequence,
A probe set in which the base sequence encoding the hairpin structure is modified to reduce base overlap interactions. In claim 1,
A probe set, wherein the first region and the second region in the target nucleic acid are located at a distance of 0 to 1 base. In claim 1,
A probe set wherein the modification is to modify a phosphodiester bond into a phosphodiester bond to reduce the base-overlapping interaction. In claim 1,
A probe set capable of polymerizing nucleic acids from the 3'-end of the hairpin structure using chain-displacement nucleic acid polymerase. In claim 1,
A probe set used to amplify genes at isotherm without using ligase. The probe set of claim 1; and
A composition for amplifying a target nucleic acid comprising a chain-displacement nucleic acid polymerase. In claim 6,
A composition for amplifying a target nucleic acid, wherein the chain displacement nucleic acid polymerase is Bst DNA polymerase. Composition for amplifying target nucleic acid of claim 6;
a gRNA that binds to at least a portion of a target nucleic acid;
Cas12 enzyme; and
A kit for detecting target nucleic acids, including a single-stranded nucleic acid probe containing a fluorescent dye and a quencher. In claim 8,
A kit for detecting a target nucleic acid, wherein the gRNA specifically binds to both part of the first region and part of the second region. A method of amplifying a target nucleic acid at isotherm using the composition of claim 6. Amplifying a target nucleic acid using the composition of claim 6; and
Generating a fluorescent signal using a single-stranded nucleic acid probe containing gRNA, Cas12, a fluorescent dye, and a quencher that binds to at least a portion of the target nucleic acid. In claim 11,
A method for detecting a target nucleic acid, further comprising: amplifying the target nucleic acid and the other target nucleic acid using the composition of claim 6, which amplifies the target nucleic acid.