Artificial nucleic acid probe capable of nucleic acid sequence-specific binding to double-stranded nucleic acid by invasion, and method for sequence-specific binding of artificial nucleic acid probe to nucleic acid sequence in double-stranded nucleic acid using the artificial nucleic acid probe

Abstract

To provide an artificial nucleic acid probe capable of nucleic acid sequence-specific binding to double-stranded nucleic acid by invasion, the probe offering enhanced flexibility in nucleic acid sequence design, and to provide a method for sequence-specific binding of an artificial nucleic acid probe to a nucleic acid sequence in a double-stranded nucleic acid using the artificial nucleic acid probe.SOLUTION: The present invention provides a method comprising a step of holding, together with a double-stranded nucleic acid, a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence, and then irradiating with light. By this method, the single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is caused to invade a double-stranded nucleic acid and to bind to a complementary sequence portion of one strand within the double-stranded nucleic acid, thereby producing a complex of the single-stranded nucleic acid containing the photoresponsive artificial nucleoside and the double-stranded nucleic acid.SELECTED DRAWING: Figure 2A

Description

[Technical Field]

[0001] The present invention relates to an artificial nucleic acid probe capable of invading double-stranded nucleic acid and binding thereto in a nucleic acid sequence-specific manner, and a method for using the artificial nucleic acid probe to bind to a nucleic acid sequence in a double-stranded nucleic acid in a sequence-specific manner. [Background technology]

[0002] One of the fundamental technologies in the field of molecular biology is the technology of artificial nucleic acid probes that specifically recognize nucleic acid sequences. This artificial nucleic acid probe technology has many applications because it enables specific recognition of nucleic acid sequences.

[0003] Therefore, artificial nucleic acid probe technology is an extremely important technology not only for basic research in molecular biology but also for use in, for example, diagnosis and treatment in the medical field, or in the development and production of therapeutic and diagnostic agents, etc., and in the development and production of enzymes, microorganisms, etc. In particular, with the recent growing expectations for tailor-made treatment, artificial nucleic acid probe technology has been attracting attention as a technology for easier genetic diagnosis.

[0004] In the specific recognition of nucleic acid sequences using such artificial nucleic acid probe technology, a technology that can specifically recognize nucleic acid sequences not in single-stranded nucleic acids but in double-stranded nucleic acids, i.e., a technology of artificial nucleic acid probes that can invade double-stranded nucleic acids to form complementary base pairs, is a particularly important technology because it is expected to be applied to antigene methods and genomic in situ hybridization (GISH).

[0005] Non-Patent Document 1 discloses a set of two artificial nucleic acids containing 3-cyanovinylcarbazole and 5-cyanouracil in the same molecule as a means for achieving such double-stranded nucleic acid invasion. According to Non-Patent Document 1, this set of artificial nucleic acids is designed to have a specific sequence structure, with each of the two artificial nucleic acids forming a complementary pair with the respective strand of the nucleic acid double strand, thereby preventing self-association between the probes and exhibiting excellent invasion ability. Non-Patent Document 1 discloses that a set of such a combination of two artificial nucleic acids functions as a probe capable of invasion into double-stranded nucleic acids.

[0006] Patent Document 1 is a photoresponsive artificial nucleotide CNV K and its manufacturing method are disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2009 / 066447

[0008] [Non-Patent Document 1] Shigetaka Nakamura, Hayato Kawabata and Kenzo Fujimoto, Chem.Commun., 2017, 53, pp7616-7619 Summary of the Invention [Problem to be solved by the invention]

[0009] From a thermodynamic point of view, it is extremely difficult for a DNA probe to invade a stable DNA double strand and form new complementary pairs with each strand of the DNA double strand. From this thermodynamic point of view, such DNA probes are usually designed as a set of double strands so that they can form as many complementary pairs as possible with each strand of the DNA double strand and become as thermodynamically stable as possible.

[0010] The probe disclosed in Non-Patent Document 1 is also designed in accordance with this common technical knowledge as a probe consisting of a set of two artificial nucleic acids, and is devised to be as thermodynamically stable as possible by each of the two artificial nucleic acids forming a complementary pair with each strand of the DNA double strand.

[0011] On the other hand, while designing a probe consisting of a set of two artificial nucleic acids is expected to be thermodynamically advantageous, it has the disadvantage that the nucleic acid sequence of each strand must be designed taking into account the nucleic acid sequence structure of the other strand, which makes the design complex, including the placement of the artificial nucleic acids to prevent self-association, and reduces the freedom of designing the nucleic acid sequence.

[0012] Therefore, an object of the present invention is to provide an artificial nucleic acid probe that can invade double-stranded nucleic acid and bind specifically to the nucleic acid sequence, which allows greater freedom in designing the nucleic acid sequence, and to provide a method for using the artificial nucleic acid probe to bind sequence-specifically to a nucleic acid sequence in a double-stranded nucleic acid. [Means for solving the problem]

[0013] The present inventors have discovered that the above object can be achieved by the artificial nucleic acid probe and a method using the artificial nucleic acid probe described below, and have arrived at the present invention.

[0014] Therefore, the present invention includes the following (1) and the following. (1) a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, A method for producing a complex of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence and a double-stranded nucleic acid by invading a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence into a double-stranded nucleic acid and binding it to the complementary sequence portion of one strand in the double-stranded nucleic acid. (2) The method according to (1), wherein the photoresponsive artificial nucleoside has a photoresponsive artificial base represented by the following formula I as a base moiety of the nucleoside: JPEG2025118414000002.jpg72170 (wherein in Formula I, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; N- represents a monovalent radical of N). (3) In a complex of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence and a double-stranded nucleic acid, a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence forms a complementary base pair with a complementary sequence portion of one strand in the double-stranded nucleic acid; The method according to (1), wherein the photoresponsive artificial nucleoside forms a photobridge and bonds with the photoresponsive artificial base and the photocrosslinkable base in the complementary sequence portion of one strand of the double-stranded nucleic acid by means of the photoresponsive artificial base that the photoresponsive artificial nucleoside has as the base portion of the nucleoside. (4) The method according to (3), wherein the photoresponsive artificial base and the photocrosslinkable base in the complementary sequence portion of one strand of the double-stranded nucleic acid are located at a position complementary to the base adjacent to the 5' end of the photoresponsive artificial base. (5) A single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is Formula II: (Formula II) JPEG2025118414000003.jpg108170 (wherein, in Formula II, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 are each independently a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen. or a ribonucleotide of the following formula III: (Formula III) JPEG2025118414000004.jpg103170 (wherein, in Formula III, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 are each independently a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen. A deoxyribonucleotide represented by the following formula IV: (Formula IV) JPEG2025118414000005.jpg124170 (wherein in Formula IV, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; Rb represents the pentose group to which the group of formula I is bonded, and the oligonucleotide chain in which the pentose group is introduced by a phosphodiester bond, among oligonucleotide chains in which a ribonucleotide or deoxyribonucleotide having a group of formula I introduced as a base moiety is introduced into the base sequence by a phosphodiester bond. The method according to (2), wherein the nucleic acid is a nucleic acid represented by the formula: (6) The method according to (1), wherein the light irradiation is light irradiation having a wavelength in the range of 360 to 400 nm. (7) The method according to (1), wherein the light irradiation is carried out for a time period ranging from 0.01 to 90 seconds. (8) In a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, The method according to (1), wherein the holding is performed at a temperature in the range of 4 to 40°C for a holding time of 10 to 120 minutes. (9) In a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, The method according to (1), wherein the retention is in a solution having a pH in the range of 5 to 8. (10) A single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, which comprises a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence, A single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, wherein the photoresponsive artificial nucleoside is a photoresponsive artificial nucleoside having a photoresponsive artificial base represented by formula I (2) as the base moiety of the nucleoside. (11) The single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding according to (10), wherein the single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is a nucleic acid represented by formula IV of (5). (12) A method for binding to a complementary sequence portion of one strand in a double-stranded nucleic acid using a single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding according to (10) or (11). [Effects of the Invention]

[0015] According to the present invention, an artificial nucleic acid probe can be realized that can invade double-stranded nucleic acid and bind specifically to a nucleic acid sequence, allowing greater freedom in nucleic acid sequence design, and this artificial nucleic acid probe can be used to bind a probe in a sequence-specific manner to a nucleic acid sequence in a double-stranded nucleic acid. According to the present invention, a probe consisting of a single artificial nucleic acid strand, rather than a probe consisting of two artificial nucleic acid strands, can be realized, and this artificial nucleic acid probe can be used to bind a probe in a sequence-specific manner to a nucleic acid sequence in a double-stranded nucleic acid. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is an explanatory diagram showing the flow of synthesis of an amidite form of the photoresponsive artificial nucleotide CNVK (Scheme 1). [Figure 2A] FIG. 2A is an explanatory diagram showing the experimental flow of photocrosslinking of long double-stranded DNA by CNVK-containing ODN in Example 2. [Figure 2B] FIG. 2B is an image of an electrophoresis gel obtained from the experiment of FIG. 2A. [Figure 2C] FIG. 2C is a graph of the photocrosslinking rate calculated from the electrophoresis gel image of FIG. 2B. [Figure 2D] FIG. 2D is an enlarged portion of the graph of FIG. 2C. [Figure 3A] FIG. 3A is an image of an electrophoresis gel obtained by an experiment in Example 3 in which ODNs with CNVK introduced at different positions were photocrosslinked to long double-stranded DNA. [Figure 3B] FIG. 3B is a graph of the photocrosslinking rate calculated from the electrophoresis gel image of FIG. 3A. [Figure 4A] FIG. 4A is an image of an electrophoresis gel obtained by an experiment on the effect of equivalent amounts of CNVK-containing ODN on photocrosslinking of long double-stranded DNA in Example 4. [Figure 4B] FIG. 4B is a graph of the photocrosslinking rate calculated from the electrophoresis gel image of FIG. 4A. [Figure 5]FIG. 5 is an image of an electrophoresis gel obtained in the experiment of Example 5 to confirm that a single-stranded probe was photocrosslinked to a long double-stranded DNA. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the specific embodiments described below.

[0018] [Formation of a complex between a single-stranded nucleic acid containing a photoresponsive artificial nucleoside and a double-stranded nucleic acid] The present invention provides a method for producing a photo-responsive artificial nucleoside-containing nucleic acid by a method comprising the steps of: holding a single-stranded nucleic acid containing a photo-responsive artificial nucleoside in its sequence together with a double-stranded nucleic acid; and then irradiating the single-stranded nucleic acid with light. The method involves invading a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence into a double-stranded nucleic acid and binding it to the complementary sequence portion of one strand in the double-stranded nucleic acid, thereby producing a complex of the single-stranded nucleic acid containing the photoresponsive artificial nucleoside in its sequence and the double-stranded nucleic acid.

[0019] In a preferred embodiment, the present invention provides a method for producing a photoreactive artificial nucleoside by a method comprising the steps of: holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid, and then irradiating the single-stranded nucleic acid with light; There is also a method in which a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is invaded into a double-stranded nucleic acid and bound to the complementary sequence portion of one strand in the double-stranded nucleic acid.

[0020] That is, according to the present invention, a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence can be invaded into a double-stranded nucleic acid and bound to the complementary sequence portion of one strand in the double-stranded nucleic acid, thereby forming a complex between the single-stranded nucleic acid containing the photoresponsive artificial nucleoside in its sequence and the double-stranded nucleic acid, and such a complex can be formed and produced.

[0021] In the common technical knowledge prior to the present invention, it was extremely difficult from a thermodynamic point of view for a DNA probe to invade a stable DNA double strand and form a new complementary pair with that DNA double strand. Therefore, such DNA probes were usually designed as a set of double strands so that they would form as many complementary pairs as possible with each strand of the DNA double strand, thereby making it as thermodynamically stable as possible.

[0022] However, the present inventors have discovered that when a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is used as a nucleic acid probe, it can invade a stable DNA double-stranded strand and form a complex of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence and a double-stranded nucleic acid without having to design and use it as a double-stranded set.

[0023] Furthermore, this invasion and complex formation occurred rapidly in a short period of time, with high yields, overturning the conventional wisdom that, from a thermodynamic standpoint, it would be disadvantageous to design nucleic acid probes as double-stranded sets.

[0024] Furthermore, since the nucleic acid probe according to the present invention is a nucleic acid probe consisting of a single-stranded nucleic acid, the sequence design of the strands is simpler than that of a nucleic acid probe designed as a set of two strands because there is no need to consider interactions between the strands, thereby maximizing the degree of freedom in designing the nucleic acid sequence.

[0025] Furthermore, since the nucleic acid probe according to the present invention is a nucleic acid probe consisting of a single-stranded nucleic acid, it is advantageous in principle compared to nucleic acid probes designed as a set of two strands in that it can avoid yield reductions and the like due to unexpected interactions that can occur even with careful consideration.

[0026] [Photoresponsive artificial nucleosides] In a preferred embodiment, the photoresponsive artificial nucleoside can have a photoresponsive artificial base represented by the following formula I as the base moiety of the nucleoside.

[0027] Formula I: JPEG2025118414000006.jpg70170

[0028] In a preferred embodiment, Ra in formula I is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, and more preferably a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group. The alkoxycarbonyl group is preferably a C2 to C7 group, more preferably a C2 to C6 group, even more preferably a C2 to C5 group, even more preferably a C2 to C4 group, still more preferably a C2 to C3 group, and particularly preferably a C2 group.

[0029] In a preferred embodiment, R1 and R2 in Formula I are each independently a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, and more preferably a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group. The alkoxycarbonyl group is preferably a C2-C7 group, more preferably a C2-C6 group, even more preferably a C2-C5 group, even more preferably a C2-C4 group, even more preferably a C2-C3 group, and particularly preferably a C2 group.

[0030] In a preferred embodiment, N- in formula I represents a monovalent group of N, and this moiety is glycosidicly bonded to position 1 of a pentose sugar, thereby introducing the artificial base, i.e., the following formula II:

[0031] JPEG2025118414000007.jpg108170

[0032] or a ribonucleotide represented by the following formula III:

[0033] JPEG2025118414000008.jpg103170

[0034] is introduced into the base sequence of the oligonucleotide via a phosphodiester bond, and is represented by the following formula IV:

[0035] JPEG2025118414000009.jpg91170

[0036] (wherein Rb represents the pentose group to which the group of formula I is bonded, and the oligonucleotide chain into which the pentose group is introduced via a phosphodiester bond, among the oligonucleotide chains in which a ribonucleotide or deoxyribonucleotide having a group of formula I introduced as a base moiety is introduced into the base sequence via a phosphodiester bond). The number of artificial bases of formula I introduced into one oligonucleotide chain is not limited to one.

[0037] [Photocrosslinking] In a preferred embodiment, photocrosslinks are formed by irradiation with light. The photocrosslinks formed by irradiation with light are covalent bonds and are sufficiently stable.

[0038] In a preferred embodiment, the formation of this photocrosslink causes the single-stranded nucleic acid invaded into the double-stranded nucleic acid to form complementary base pairs with a strand having a complementary base sequence, and this state is fixed by the photocrosslink, resulting in a thermodynamically stable state.

[0039] Photocrosslinking is formed between a single-stranded nucleic acid containing a photoresponsive artificial nucleoside and one of the two strands in a double-stranded nucleic acid that has a base sequence complementary to the single-stranded nucleic acid containing the photoresponsive artificial nucleoside.

[0040] More specifically, photocrosslinking is formed between a photoresponsive artificial base in a single-stranded nucleic acid containing a photoresponsive artificial nucleoside and a photoresponsive artificial base and a photocrosslinkable base in one of the two strands in a double-stranded nucleic acid that has a base sequence complementary to the single-stranded nucleic acid containing the photoresponsive artificial nucleoside.

[0041] In a preferred embodiment, the photoresponsive artificial base and the photocrosslinkable base in the complementary sequence portion of one strand of the double-stranded nucleic acid are located at a position complementary to the base adjacent to the 5' end of the photoresponsive artificial base.

[0042] In a preferred embodiment, the photoresponsive artificial base and the photocrosslinkable base are bases having a pyrimidine ring. That is, the photoresponsive artificial base and the photocrosslinkable base are, for example, cytosine, uracil, and thymine, furthermore pseudouracil and pseudothymine, preferably thymine and cytosine, particularly preferably thymine. On the other hand, the photoresponsive artificial base and the photocrosslinkable base are not bases having a purine ring. Taking these into consideration, those skilled in the art can design the nucleic acid base sequence of the single-stranded nucleic acid probe according to the present invention.

[0043] In a preferred embodiment, there are no particular restrictions on the base at the position that forms a base pair with the photoresponsive artificial base in the nucleic acid base sequence to which the single-stranded nucleic acid probe hybridizes, and any base may be used.

[0044] [Light irradiation] In a preferred embodiment, light irradiation for forming photocrosslinks can be performed using light having a wavelength in the range of 360 to 400 nm, preferably 375 to 400 nm, or light having a wavelength of 385 nm, particularly preferably laser light having a single wavelength of 385 nm.

[0045] In a preferred embodiment, an extremely short time is sufficient for the light irradiation time, for example, 0.01 to 90 seconds, 0.01 to 75 seconds, 0.01 to 60 seconds, 0.01 to 30 seconds, 0.01 to 20 seconds, 0.01 to 10 seconds, 0.01 to 5 seconds, 0.01 to 2 seconds, 0.01 to 1 second, 0.05 to 90 seconds, 0.05 to 75 seconds, 0.05 to 60 seconds, 0.05 to 30 seconds, 0.05 to 20 seconds, 0.05 to 10 seconds, 0.05 to 5 seconds The light irradiation time can be set to 0.05 to 2 seconds, 0.05 to 1 second, 0.1 to 90 seconds, 0.1 to 75 seconds, 0.1 to 60 seconds, 0.1 to 30 seconds, 0.1 to 20 seconds, 0.1 to 10 seconds, 0.1 to 5 seconds, 0.1 to 2 seconds, 0.1 to 1 second, 0.2 to 90 seconds, 0.2 to 75 seconds, 0.2 to 60 seconds, 0.2 to 30 seconds, 0.2 to 20 seconds, 0.2 to 10 seconds, 0.2 to 5 seconds, 0.2 to 2 seconds, or 0.2 to 1 second.

[0046] Prior to photoirradiation, the single-stranded nucleic acid is held together with the double-stranded nucleic acid. This photocrosslinking is advantageous in that there are no particular restrictions on the pH, salt concentration, etc. during photoirradiation because it uses a photoreaction. Therefore, in a preferred embodiment, the conditions for photoirradiation, such as temperature, pH, and salt concentration, can be the same as those for the holding performed immediately before.

[0047] [Keep] Prior to light irradiation, the single-stranded nucleic acid is maintained together with the double-stranded nucleic acid.

[0048] In a preferred embodiment, the storage can be carried out at a temperature in the range of, for example, 4 to 40° C., or 15 to 40° C., or 25 to 40° C. In a preferred embodiment, the storage can be carried out under the same temperature conditions as in vivo, for example, at a temperature in the range of 35 to 40° C.

[0049] In a preferred embodiment, the holding time can be, for example, in the range of 10 to 120 minutes, or in the range of 10 to 60 minutes, or in the range of 10 to 30 minutes.

[0050] In a preferred embodiment, the retention can be carried out in a solution having a pH range of, for example, 5-8, or 6-8, or 6.5-8.

[0051] In a preferred embodiment, the retention is not particularly limited as long as the invaded single-stranded nucleic acid is retained in a solution in which it can form complementary base pairs with a strand of the double-stranded nucleic acid prior to photocrosslinking and remain photocrosslinkable. For example, the retention can be carried out in a solution having a salt concentration of physiological saline, such as a salt concentration of a physiological buffer solution, for example, a salt concentration of 0 mM to 300 mM NaCl.

[0052] [Single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding] The present invention also relates to a single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, which is composed of a single-stranded nucleic acid used in the above-mentioned method for producing a complex of a single-stranded nucleic acid and a double-stranded nucleic acid. This single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding is composed of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence, and the photoresponsive artificial nucleoside has the photoresponsive artificial base represented by the above formula I as the base moiety of the nucleoside.

[0053] Furthermore, the present invention also relates to a method for binding to a complementary sequence portion of one strand in a double-stranded nucleic acid using a single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding.

[0054] By binding to the complementary sequence portion of one strand in such a double-stranded nucleic acid, it is possible to detect the nucleic acid base sequence in the double-stranded nucleic acid and inhibit transcription or expression. Therefore, the present invention also relates to a method for detecting the nucleic acid base sequence in a double-stranded nucleic acid using a single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, and to a method for inhibiting the transcription or expression of a gene corresponding to the double-stranded nucleic acid.

[0055] [Single-stranded nucleic acid chain length] In a preferred embodiment, the chain length of a single-stranded nucleic acid that can be used as a single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding is not particularly limited as long as it is within a range that allows the desired invasion to be carried out, and can be, for example, a chain length in the range of 5 to 100 bases, 8 to 80 bases, 10 to 60 bases, 10 to 40 bases, or 10 to 30 bases. [Example]

[0056] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples illustrated below.

[0057] [Example 1] [ CNV Synthesis of K-containing oligonucleotides] The following formula V:

[0058] JPEG2025118414000010.jpg101170

[0059] Nucleotides represented by ( CNV In order to produce an ODN (oligodeoxyribonucleotide) having the base sequence (K), synthesis was carried out according to Scheme 1 in Figure 1. The synthesis was carried out according to the procedure disclosed in Patent Document 1 (International Publication No. WO2009 / 066447).

[0060] The amidite form of the photoresponsive artificial nucleotide 3-cyanovinylcarbazole nucleotide synthesized as described above was used for DNA synthesis using a DNA synthesizer described below.

[0061] The oligonucleotides (ODN) used in the following experiments were synthesized using a DNA synthesizer (NTS M-2-MIX_NPS) with a 1.0 μmol scale High Load-CPG (Glen Research). The synthesized oligonucleotides were cleaved from the CPG with 28% aqueous ammonia and deprotected by incubation at 65°C for 4 hours. The deprotected oligonucleotides were purified by HPLC using a 2-40% acetonitrile gradient at a flow rate of 1 ml / min. Target DNA was purchased from Integrated DNA Technologies Co.

[0062] The sequences of the synthesized oligonucleotides (DI probes, Duplex Invasion Probes) are shown in Table 1. The sequences of the target DNAs are shown in Table 2. The target DNAs were prepared as double-stranded DNAs containing the sequences in Table 2 and used in the experiments described below.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Example 2] [ CNV Photocrosslinking of long double-stranded DNA by K-containing ODN] 2.5 nM target DNA was mixed with 10 mM Tris HCl buffer (pH 7.4) and incubated at 37°C for 15 minutes, after which each probe was added at 2.5 μM. The target DNA was added in a double-stranded state. After incubation at 37°C for 1 hour, the probe was measured using an Omunicure at 385 nm (12400 mW / cm 2The reaction mixture was irradiated with light for 0, 0.1, 0.2, 0.5, 1, 10, and 60 seconds at 1000 kJ / min. A denaturant (saturated urea in formamide) was added to the reaction mixture, and the mixture was incubated at 90°C for 5 minutes. The sample solution was analyzed by denaturing PAGE using an 8% polyacrylamide gel. Images of Cy3 and SYBR Gold were captured using an LAS-3000, and band intensities were determined using ImageJ.

[0066] An explanatory diagram showing the flow of this experiment is shown in Figure 2A. The base sequence in the partially enlarged view of the long double-stranded DNA shown in Figure 2A is a partially enlarged version of the base sequence near the center of the Target DNA in Table 2, so the base sequence used in Figure 2A is exactly the same as the base sequence disclosed as the Target DNA in Table 2.

[0067] An image of the electrophoresis gel obtained from this experiment is shown in Figure 2B.

[0068] The photocrosslinking rate was calculated from the band intensity obtained in this experiment. A photocrosslinking rate of 100% means that all molecules of the probe introduced were photocrosslinked. A graph of the calculated photocrosslinking rate is shown in Figure 2C. The horizontal axis of Figure 2C represents the light irradiation time (seconds), and the vertical axis represents the photocrosslinking rate (%).

[0069] As shown in FIG. 2C, this photocrosslinking reaction was rapid, reaching a photocrosslinking rate of approximately 80% in just 10 seconds.

[0070] An enlarged portion of Figure 2C is shown in Figure 2D, which is a graph showing an enlarged view of the change from 0 seconds to 1 second on the horizontal axis of the graph in Figure 2C.

[0071] As shown in FIG. 2D, this photocrosslinking reaction was extremely rapid, reaching a photocrosslinking rate of approximately 60% in just 0.5 seconds.

[0072] The results of denaturing PAGE analysis confirmed that photocrosslinked bands appeared in lanes irradiated with light from Lane 3 onwards. The crosslinking rate was approximately 60% at 0.5 seconds, and an 80% yield of photocrosslinked products was confirmed over 60 seconds.

[0073] Furthermore, in a separate experiment, it was found that, in an experiment similar to the above experiment, the artificial nucleic acid probe of the present invention was used to incubate at 37°C for 1 hour and then irradiate with 385 nm light. Instead of the 1 hour incubation time that had been set as a sufficient incubation time, incubation times of 0, 15, 30, 45, and 60 minutes were used, followed by 60 seconds of irradiating with 385 nm light. As a result, the photocrosslinking rate exceeded 10% in 15 minutes, exceeded 40% in 30 minutes, exceeded 55% in 45 minutes, and exceeded 65% in 60 minutes. This indicates that incubation times shorter than 60 minutes are sufficient for the practical detection of probe binding and the resulting double-stranded sequence.

[0074] [Example 3] [ CNV Photocrosslinking of long double-stranded DNA by ODNs with different K-introduction sites CNV To verify the change in photocrosslinking efficiency and sequence selectivity depending on the K-introduction position, similar photocrosslinking experiments were performed using four types of probes (5'K probe, midK probe, 3'K probe, and GK probe). The double strand of target DNA was used as the long double-stranded DNA. 2.5 nM target DNA was mixed with 10 mM Tris HCl buffer (pH 7.4) and incubated at 37°C for 15 minutes, after which each probe was added at 2.5 μM. After incubation at 37°C for 1 hour, the samples were analyzed using an Omunicure at 385 nm (12,400 mW / cm). 2The reaction mixture was irradiated with light for 60 seconds at 90°C. A denaturant (saturated urea in formamide) was added to the reaction mixture, and the mixture was denatured at 90°C for 5 minutes. The sample solution was analyzed by 8% denaturing PAGE. The SYBR Gold denaturing PAGE results were photographed using an LAS-3000, and band intensities were quantified using ImageJ, a quantitative analysis software.

[0075] An image of the electrophoresis gel obtained from this experiment is shown in Figure 3A.

[0076] The photocrosslinking rate was calculated from the band intensity obtained in this experiment. A photocrosslinking rate of 100% means that all molecules of the probe introduced were photocrosslinked. A graph of the calculated photocrosslinking rate is shown in Figure 3B. The horizontal axis of Figure 3B indicates the probe classification used, and the vertical axis is the photocrosslinking rate (%).

[0077] From the results of this experiment, CNV It was found that changing the position of K had almost no effect on the photocrosslinking efficiency. Furthermore, when the photocrosslinking position was replaced with guanine, no photocrosslinked band was observed, confirming high sequence selectivity.

[0078] [Example 4] [ CNV Effect of equivalent number of K-containing ODN on photocrosslinking of long double-stranded DNA CNVTo verify the effect of the equivalent amount of K-containing ODN on photocrosslinking of long double-stranded DNA, a similar photocrosslinking experiment was performed using a probe (midK Probe). The long double-stranded DNA was a double-stranded target DNA. 2.5 nM target DNA was mixed with 10 mM Tris HCl buffer (pH 7.4) and incubated at 37°C for 15 minutes. Each probe was added at concentrations of 25, 50, 125, 250, or 2500 nM. The mixture was incubated at 37°C for 1 hour and then irradiated with light at 385 nm for 60 seconds using an Omunicure. A denaturant (saturated urea in formamide) was added to the reaction solution, and the mixture was heat-denatured at 90°C for 5 minutes. The sample solution was analyzed by 8% denaturing PAGE. SYBR Gold denaturing PAGE images were captured using a LAS-3000, and band intensities were quantified using ImageJ quantitative analysis software.

[0079] An image of the electrophoresis gel obtained from this experiment is shown in Figure 4A.

[0080] The photocrosslinking rate was calculated from the band intensity obtained in this experiment. A photocrosslinking rate of 100% means that all molecules of the probe added were photocrosslinked. A graph of the calculated photocrosslinking rate is shown in Figure 4B. The horizontal axis of Figure 4B shows the amount of probe used (number of equivalents), and the vertical axis shows the photocrosslinking rate (%). One equivalent of the probe used means that one molecule of probe is used per one molecule of double-stranded target DNA molecule.

[0081] Even at 10 equivalents, a band of approximately 10% photocrosslinking was observed, and at 100 equivalents, a photocrosslinking efficiency of 50% was confirmed, and at 1000 equivalents, a photocrosslinking efficiency of 70% was confirmed. This suggests that the photocrosslinking efficiency can be adjusted by changing the number of equivalents.

[0082] [Example 5] [Confirmation of photocrosslinking of single-stranded probe to long double-stranded DNA] Further confirmation of photocrosslinking of single-stranded probes to long double-stranded DNA was carried out by the following experiment. The double strands of target DNA were used as the long double-stranded DNA. Probes complementary to the sense and antisense strands of target DNA were prepared, and the photocrosslinking efficiency was confirmed by changing the concentration ratio. The sequences of the probes (DI probe and DDI probe) used (Duplex Invasion Probe, Double Duplex Invasion Probe) are shown in Table 3.

[0083] [Table 3]

[0084] Probes 1 and 2 in Table 3 have complementary sequences that can form a double strand by shifting the five bases at the ends. Therefore, when probes 1 and 2 coexist, they form a double strand and are a combination that can act as a double-stranded artificial nucleic acid probe (DDI probe, Double Duplex Invasion Probe) that can invade double-stranded nucleic acids and bind specifically to nucleic acid sequences.

[0085] Probe 1 and probe 2 in Table 3 are a combination that can act alone as a single-stranded artificial nucleic acid probe (DI probe, Duplex Invasion Probe) that can invade double-stranded nucleic acid and bind specifically to the nucleic acid sequence.

[0086] Used for probe 1 and probe 2 in Table 3 CN U is a 5-cyanouracil nucleotide, which was used for DNA synthesis in a DNA synthesizer after forming an amidite similar to that of 3-cyanovinylcarbazole nucleotide.

[0087] JPEG2025118414000014.jpg103170

[0088] 2.5 nM target DNA was mixed with 10 mM Tris HCl buffer (pH 7.4) and incubated at 37°C for 15 minutes, after which each probe was added at 0-5 μM. The mixture was incubated at 37°C for 1 hour and then irradiated with light at 385 nm for 60 seconds using an Omunicure. A denaturant (saturated urea in formamide) was added to the reaction solution, and the mixture was heat-denatured at 90°C for 5 minutes. The sample solution was analyzed by 8% denaturing PAGE. SYBR Gold denaturing PAGE results were photographed using an LAS-3000, and band intensity was quantified using ImageJ quantitative analysis software.

[0089] An image of the electrophoresis gel obtained from this experiment is shown in FIG.

[0090] In the electrophoresis gel image shown in Figure 5, two bands derived from the photocrosslinked product were confirmed in Lanes 5 to 9, which contained two types of probes. However, bands derived from the photocrosslinked product also appeared in Lanes 3 and 11, which contained only one probe. According to previous studies by the present inventors, CVN K and CN It was concluded that only by combining U can DDI photocrosslinking occur by invading the target double-stranded DNA while preventing self-association. However, the present invention suggests photocrosslinking using a single type of probe, CNV It was found that by using only one type of probe incorporating only K, the target DNA double strand can be invaded, and the probe sequence can form a complementary pair with the target nucleic acid sequence, resulting in photocrosslinking. [Industrial Applicability]

[0091] The present invention provides an artificial nucleic acid probe that can invade double-stranded nucleic acid and bind to it in a nucleic acid sequence-specific manner, allowing greater freedom in designing the nucleic acid sequence, and a method for sequence-specifically binding the artificial nucleic acid probe to a nucleic acid sequence in a double-stranded nucleic acid using the artificial nucleic acid probe. The present invention is an industrially useful invention.

Claims

1. a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, A method for producing a complex of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence and a double-stranded nucleic acid by invading a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence into a double-stranded nucleic acid and binding it to the complementary sequence portion of one strand in the double-stranded nucleic acid.

2. The method according to claim 1, wherein the photoresponsive artificial nucleoside has a photoresponsive artificial base represented by the following formula I as a base moiety of the nucleoside: (However, in Formula I, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; N- represents a monovalent radical of N).

3. In a complex of a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence and a double-stranded nucleic acid, a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence forms a complementary base pair with a complementary sequence portion of one strand in the double-stranded nucleic acid; The method of claim 1, wherein the photoresponsive artificial nucleoside forms a photobridge and bonds with the photoresponsive artificial base and a photocrosslinkable base in the complementary sequence portion of one strand of the double-stranded nucleic acid via the photoresponsive artificial base that the photoresponsive artificial nucleoside has as the base portion of the nucleoside.

4. The method according to claim 3, wherein the photoresponsive artificial base and the photocrosslinkable base in the complementary sequence portion of one strand of the double-stranded nucleic acid are located at a position complementary to the base adjacent to the 5' end of the photoresponsive artificial base.

5. A single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence, The following formula II: (Formula II) (However, in Formula II, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 are each independently a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen. or a ribonucleotide represented by the following formula III: (Formula III) (However, in Formula III, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 are each independently a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen. A deoxyribonucleotide represented by the following formula IV: (Formula IV) (However, in Formula IV, Ra is a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; R1 and R2 each independently represent a cyano group, an amide group, a carboxyl group, a C2 to C7 alkoxycarbonyl group, or hydrogen; Rb represents the pentose group to which the group of formula I is bonded, and the oligonucleotide chain into which the pentose group is introduced by a phosphodiester bond, among oligonucleotide chains in which a ribonucleotide or deoxyribonucleotide having a group of formula I introduced as a base moiety is introduced into the base sequence by a phosphodiester bond. The method according to claim 2, wherein the nucleic acid is represented by the formula:

6. The method according to claim 1, wherein the light irradiation is light irradiation with light having a wavelength in the range of 360 to 400 nm.

7. The method according to claim 1, wherein the light irradiation is carried out for a time period ranging from 0.01 to 90 seconds.

8. In a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, 2. The method of claim 1, wherein the holding is at a temperature in the range of 4 to 40°C for a holding time of 10 to 120 minutes.

9. In a step of holding a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence together with a double-stranded nucleic acid and then irradiating the single-stranded nucleic acid with light, 2. The method of claim 1, wherein the retaining is in a solution having a pH in the range of 5 to 8.

10. A single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, which comprises a single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence, A single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding, wherein the photoresponsive artificial nucleoside is a photoresponsive artificial nucleoside having a photoresponsive artificial base represented by formula I of claim 2 as the base moiety of the nucleoside.

11. The single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding described in claim 10, wherein the single-stranded nucleic acid containing a photoresponsive artificial nucleoside in its sequence is a nucleic acid represented by formula IV of claim 5.

12. A method for binding to a complementary sequence portion of one strand in a double-stranded nucleic acid using the single-stranded artificial nucleic acid probe for double-stranded nucleic acid invasion binding according to claim 10 or 11.

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