Novel uses of staple nucleic acids

Abstract

To develop various applications of Staple nucleic acids.SOLUTION: The inventors of the present invention have demonstrated that Staple nucleic acids developed to date form a G-quadruplex structure on a target DNA through a total of four repeated guanine sequences, including repeated guanine sequences on the target DNA, thereby enabling alteration of the structure of the target DNA. As the result of such an effect, the inventors have developed novel applications of Staple nucleic acids (e.g. regulation of transcription from DNA to RNA), thereby solving the above problem.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel use of Staple nucleic acids.

Background Art

[0002] As gene expression suppression techniques, various tools such as microRNA and siRNA are known (Non-Patent Document 1, Non-Patent Document 2). If the sequence information of a target gene is known, siRNA can be designed relatively easily. By referring to existing reports on the target gene and selecting multiple target sequences for one target mRNA, gene expression can be strongly suppressed.

[0003] Regarding any of these techniques, since the gene expression suppression function is exerted by a simple mechanism of binding to mRNA, an unexpected effect (off-target effect) cannot be avoided (Non-Patent Document 3), and it contains problems that must be solved for use in the human body.

[0004] With the current improvement in bioinformatics technology, it has become possible to minimize the suppression of the expression of unintended genes (off-target effect). However, from the perspective of pharmaceutical development, siRNA development is not progressing smoothly. The reason is that nucleic acid drugs need to use modified nucleic acids. When using modified nucleic acids, it is known that problems such as a significant decrease in microRNA effect and siRNA activity, and the inability to obtain the expected protein translation suppression effect also occur (Non-Patent Document 4).

[0005] To solve these problems, various artificially modified nucleic acids have been synthesized, but the development of artificially modified nucleic acids with high versatility as pharmaceuticals is still ongoing. The biggest reason that makes the development of artificially modified nucleic acids difficult is that siRNA requires linkage with an enzymatic reaction having a cleavage activity for a target mRNA sequence. That is, while nuclease resistance is acquired by chemically modifying nucleic acids, the recognition ability as a substrate for the enzymatic reaction is lost.

[0006] To solve such problems, the inventors of the present invention have heretofore developed a Staple nucleic acid having a function of specifically binding to a target mRNA sequence in a sequence-specific manner and inducing the formation of a guanine quadruplex structure by using a guanine repeat sequence present on the target mRNA, and have clarified that the Staple nucleic acid suppresses protein expression based on the guanine quadruplex structure induced by the Staple nucleic acid (Patent Document 1).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0009] An object of the present invention is to develop various uses of Staple nucleic acids. [Means for Solving the Problems]

[0010] The inventors of the present invention have shown that the Staple nucleic acid developed so far can form a guanine quadruplex structure on the target DNA by a total of four guanine repeat sequences including the guanine repeat sequence on the target DNA, and can change the structure of the target DNA. As a result of such an action, the above problems have been solved by developing a novel use of the Staple nucleic acid (such as transcriptional regulation from DNA to RNA).

[0011] More specifically, the present application provides the following aspects in order to solve the above-described problems: [1]: A first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence portion near the guanine repeat sequence with respect to a nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, An oligonucleotide comprising: A method of changing the three-dimensional structure of a target DNA by hybridization of the oligonucleotide to the target DNA of the first nucleotide sequence and the second nucleotide sequence, shortening the spatial distance of a total of four guanine repeat sequences of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, and forming a guanine quadruplex structure by the four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA and changing the structure of the target DNA; [2]: The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA ·A nucleotide sequence portion on the 5' side or 3' side of the 3'-terminal guanine repeat sequence · The nucleotide sequence portion on the 5'-side or 3'-side of the guanine repeat sequence on the 5'-end side A method for changing the structure of the target DNA according to [1], which hybridizes to [3]: The method for changing the structure of the target DNA according to [1] or [2], wherein the oligonucleotide is composed of DNA, RNA, a modified nucleic acid, or a combination thereof; [4]: With respect to the nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence in the vicinity of the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising The oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of a total of four guanine repeat sequences of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forms a guanine quartet structure by the four guanine repeat sequences, forms a loop portion in a part of the target DNA, and inhibits the binding of RNA polymerase to the target DNA or inhibits the function of RNA polymerase, thereby regulating the transcription of RNA from the target DNA; [5]: The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA · The nucleotide sequence portion on the 5'-side or 3'-side of the guanine repeat sequence on the 3'-end side · The nucleotide sequence portion on the 5'-side or 3'-side of the guanine repeat sequence on the 5'-end side A method for regulating the transcription of RNA from the target DNA according to [3], which hybridizes to [6]: The method for regulating the transcription of RNA from the target DNA according to [3] or [4], wherein the oligonucleotide is composed of DNA, RNA, a modified nucleic acid, or a combination thereof; [7]: A nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence near the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising: A method for regulating protein expression from a target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of a total of four guanine repeat sequences of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forms a guanine quadruplex structure by the four guanine repeat sequences, forms a loop portion in a part of the target DNA, inhibits the binding of RNA polymerase to the target DNA or inhibits the function of RNA polymerase, and regulates the transcription of RNA from the target DNA; [8]: The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are · A nucleotide sequence portion on the 5'-side or 3'-side of the guanine repeat sequence on the 3'-terminal side, · A nucleotide sequence portion on the 5'-side or 3'-side of the guanine repeat sequence on the 5'-terminal side The method for regulating protein expression from a target DNA according to [7], which hybridizes to; [9]: The method for regulating protein expression from a target DNA according to [7] or [8], wherein the oligonucleotide is DNA, RNA, a modified nucleic acid, or a combination thereof;

[10] : A nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence near the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising: A method for suppressing replication of a target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridizing to the target DNA of the first nucleotide sequence and the second nucleotide sequence, and the spatial distance of four guanine repeat sequences in total, which is the combination of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, becomes shorter, and a loop portion is formed in a part of the target DNA by forming a guanine quadruplex structure by these four guanine repeat sequences, thereby inhibiting the binding of DNA polymerase to the target DNA or inhibiting the function of DNA polymerase;

[11] : The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA · The nucleotide sequence part on the 5'-side or 3'-side of the guanine repeat sequence on the 3'-terminal side, · The nucleotide sequence part on the 5'-side or 3'-side of the guanine repeat sequence on the 5'-terminal side The method for suppressing replication of a target DNA according to

[10] , which hybridizes to;

[12] : The method for suppressing replication of a target DNA according to

[10] or

[11] , wherein the oligonucleotide is DNA, RNA, a modified nucleic acid, or a combination thereof. [Advantages of the Invention]

[0012] The present invention shows that the previously developed Staple nucleic acid can form a guanine quadruplex structure on the target DNA with a total of four guanine repeat sequences including the guanine repeat sequence on the target DNA, and can change the structure of the target DNA. As a result of such an action, a novel use of the Staple nucleic acid (such as regulation of transcription from DNA to RNA) can be provided. [Brief Description of the Drawings]

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0014] The terms used in the present invention are defined as follows: (a) Staple nucleic acid: An oligonucleotide that hybridizes sequence-specifically to two sequences on a target nucleic acid, changes the three-dimensional structure of the target nucleic acid, and has a function of inducing the formation of a guanine quadruplex structure by utilizing a guanine repeat sequence present on the target nucleic acid. There are a non-G-supplying Staple nucleic acid (also referred to as a non-G-supplying oligonucleotide or a first-generation Staple nucleic acid) and a G-supplying Staple nucleic acid (also referred to as a G-supplying oligonucleotide or a second-generation Staple nucleic acid), which will be described later; (b) Non-G-supplying Staple nucleic acid: One type of Staple nucleic acid, which is prepared with reference to the Staple nucleic acid disclosed in Patent Document 1 and does not contain a guanine repeat sequence in the Staple nucleic acid itself. It refers to an oligonucleotide that hybridizes sequence-specifically to two sequences on a target nucleic acid, changes the three-dimensional structure of the target nucleic acid, and has a function of shortening the spatial distance of four guanine repeat sequences present on the target nucleic acid to induce the formation of a guanine quadruplex structure. The non-G-supplying Staple nucleic acid that can be used in the present invention is also referred to as a non-G-supplying oligonucleotide or a first-generation Staple nucleic acid; (c) G-supplied Staple nucleic acid: A type of Staple nucleic acid that contains at least one guanine repeat sequence within the Staple nucleic acid itself. It hybridizes sequence-specifically to two sequences on the target nucleic acid, changes the three-dimensional structure of the target nucleic acid, and shortens the spatial distance between a total of four guanine repeat sequences, namely the guanine repeat sequence present on the target nucleic acid and the guanine repeat sequences present in the G-supplied Staple nucleic acid, to induce the formation of a guanine quadruplex structure. The G-supplied Staple nucleic acid that can be used in the present invention is also referred to as a G-supplied oligonucleotide or a second-generation Staple nucleic acid. (d) Nucleic acid: The nucleic acid that constitutes the Staple nucleic acid that can be used in the present invention refers to either a naturally occurring nucleic acid or a nucleic acid that does not naturally exist (unnatural nucleic acid). A naturally occurring nucleic acid refers to DNA or RNA composed of nucleobases (i.e., adenine (a), guanine (g), cytosine (c), thymine (t), uracil (u)). A nucleic acid that does not naturally exist (unnatural nucleic acid) refers to a nucleic acid whose physical properties have been changed by modifying the nucleobase, sugar, and phosphodiester moieties, and is also referred to as a modified nucleic acid.

[0015] Structure of the first-generation Staple nucleic acid The first-generation Staple nucleic acid that can be used in the present invention refers to a Staple nucleic acid prepared with reference to the Staple nucleic acid disclosed in Patent Document 1. That is, specifically, this first-generation Staple nucleic acid is structurally characterized by not containing a guanine repeat sequence that constitutes a guanine quadruplex structure within the Staple nucleic acid itself.

[0016] The first-generation Staple nucleic acid that can be used in the present invention is a non-G-supplying oligonucleotide comprising a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence portions near any two of the guanine repeat sequences (for example, on the 5'- or 3'-side) of the guanine repeat sequences in the nucleotide sequence portion containing four guanine repeat sequences on the target nucleic acid, wherein the non-G-supplying oligonucleotide changes the three-dimensional structure of the target nucleic acid by hybridizing to the first nucleotide sequence and the second nucleotide sequence, shortens the spatial distance of the four guanine repeat sequences on the target nucleic acid, and has the function of forming a guanine quadruplex structure by these four guanine repeat sequences. The non-G-supplying oligonucleotide is referred to as such.

[0017] Generally, the guanine quadruplex structure in cells is a stable higher-order nucleic acid structure constructed when four guanine repeat sequences of three or more bases are present in a close spatial arrangement of less than seven bases. The inventors of the present invention have found that even guanine repeat sequences present at positions spaced apart by a spacing of seven or more bases on the target nucleic acid can be artificially spatially approximated and an artificial guanine quadruplex structure can be induced in the target nucleic acid by using the non-G-supplying oligonucleotide comprising the first nucleotide sequence and the second nucleotide sequence of the present invention designed appropriately.

[0018] In the present invention, the guanine repeat sequence in the target nucleic acid to which the first-generation Staple nucleic acid binds refers to a continuous sequence containing three or more guanines present on the target nucleic acid or a sequence containing three or more guanines including a nucleotide sequence forming a bulge structure between guanine and guanine. Specific guanine repeat sequences include, for example, GGG, GNGG (N may be any base and the number may be one or more), etc., which are guanine repeat sequences that can be elements for forming a guanine quadruplex structure reported in the literature.

[0019] For the first-generation Staple nucleic acid of the present invention to form a guanine quadruplex structure intracellularly, the oligonucleotide needs to have · the ability to recognize the sequence of the target DNA, and · the ability to induce the formation of a guanine quadruplex structure simultaneously.

[0020] The ability of the first-generation Staple nucleic acid to recognize the sequence of the target DNA is realized by the oligonucleotide, which is the first-generation Staple nucleic acid, containing a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence in the vicinity (for example, the 5'-side or 3'-side) of the guanine repeat sequence on the target DNA.

[0021] In the present invention, the sequences of the first nucleotide sequence and the second nucleotide sequence in the first-generation Staple nucleic acid are different for each target DNA sequence. Two sequence portions on the target DNA are selected so that the spatial distance of the four guanine repeat sequences on the target DNA can be shortened, and the first nucleotide sequence and the second nucleotide sequence can be determined to hybridize to these two sequence portions.

[0022] The first nucleotide sequence and the second nucleotide sequence of the first-generation Staple nucleic acid can be designed as appropriate. For example, the first nucleotide sequence and the second nucleotide sequence of the first-generation Staple nucleic acid may be selected to hybridize to · the nucleotide sequence portion in the vicinity (for example, the 5'-side or 3'-side) of the guanine repeat sequence closest to the 3'-end side among the guanine repeat sequences on the target DNA, · the nucleotide sequence portion in the vicinity (for example, the 5'-side or 3'-side) of the guanine repeat sequence closest to the 5'-end side among the guanine repeat sequences on the target DNA.

[0023] For example, but not limited to, as an example of the first-generation Staple nucleic acid of the present invention, ​· The nucleotide sequence on the downstream side (3' side) of one of the guanine repeat sequences of the target DNA (for example, a region of 13 to 20 bases starting from the bases within 20 bases on the immediate downstream side (3' side) (for example, the 1st to 20th bases on the downstream side of the guanine repeat sequence)), or · The nucleotide sequence on the upstream side (5' side) of one of the guanine repeat sequences of the target DNA (for example, a nucleotide sequence (for example, a region of 13 to 20 bases on the upstream side (5' side)) starting from the bases within 20 bases on the immediate upstream side (5' side) (for example, the 1st to 20th bases on the downstream side of the guanine repeat sequence)), etc. can be selected, and the first nucleotide sequence and the second nucleotide sequence can be designed to hybridize with those regions.

[0024] The first nucleotide sequence and the second nucleotide sequence of the first-generation Staple nucleic acid of the present invention can be appropriately designed according to their uses. For example, the following characteristics (however, not limited thereto): · At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes to a nucleotide sequence portion near the guanine repeat sequence existing before the stop codon on the target DNA; · At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes to a nucleotide sequence portion near the guanine repeat sequence existing in the 5' untranslated region on the target DNA; · The first nucleotide sequence and the second nucleotide sequence both hybridize to a nucleotide sequence portion near the guanine repeat sequence existing in the 5' untranslated region on the target DNA; · The first nucleotide sequence and the second nucleotide sequence both hybridize to a nucleotide sequence portion near the guanine repeat sequence existing in the 3' untranslated region; They can be appropriately designed to have the above characteristics, but when used for other purposes, they should be appropriately designed according to the purposes.

[0025] The distance of the nucleotide sequence region on the target DNA where the first nucleotide sequence and the second nucleotide sequence hybridize from the guanine repeat sequence of the target DNA (the number of nucleotides sandwiched between the nucleotide at the proximal end closest to the guanine repeat sequence of the first nucleotide sequence or the second nucleotide sequence and the G of the guanine repeat sequence) can be appropriately determined by calculations related to thermodynamic stability, and the distance (number of nucleotides) is not particularly limited.

[0026] Therefore, as the starting point of the region where the Staple nucleic acid hybridizes, preferably bases within 20 bases (for example, the 1st to 20th bases) from immediately downstream (3' side) or immediately upstream (5' side) of the guanine repeat sequence, more preferably bases within 15 bases (for example, the 1st to 15th bases) from immediately downstream (3' side) or immediately upstream (5' side) of the guanine repeat sequence, still more preferably bases within 10 bases (for example, the 1st to 10th bases) from immediately downstream (3' side) or immediately upstream (5' side) of the guanine repeat sequence, even more preferably bases within 5 bases (for example, the 1st to 5th bases) from immediately downstream (3' side) or immediately upstream (5' side) of the guanine repeat sequence, and even more preferably bases within 3 bases (for example, the 1st to 3rd bases) from immediately downstream (3' side) or immediately upstream (5' side) of the guanine repeat sequence can be selected.

[0027] The first-generation Staple nucleic acid can be composed of DNA, RNA, modified nucleic acids, or combinations thereof. The first-generation Staple nucleic acid of the present invention is one in which the first nucleotide sequence and the second nucleotide sequence are directly or indirectly linked via a linker. In the present invention, in each region of the first nucleotide sequence, the second nucleotide sequence, and optionally the linker that may be included, a preferred nucleic acid form can be selected.

[0028] Here, in the first-generation Staple nucleic acid, when referring to the linker that connects the first nucleotide sequence and the second nucleotide sequence, the number of bases present between the first nucleotide sequence and the second nucleotide sequence refers to the linker of any nucleic acid. In terms of the binding of the first nucleotide sequence to the target DNA and the binding of the second nucleotide sequence to the target DNA, it is necessary that the first-generation Staple nucleic acid be of a length such that it can change the three-dimensional structure of the target DNA, thereby shortening the spatial distance of the four guanine repeat sequences on the target DNA.

[0029] The first-generation Staple nucleic acid has the first nucleotide sequence and the second nucleotide sequence directly or indirectly linked via a linker. The overall length of the Staple nucleic acid varies depending on the nucleotide sequence of the target DNA and can be of any length designed based on the generally known hybridization criteria in the art, such as GC content, Tm value, etc. It can be of a nucleotide length longer or shorter than that according to its design. As an example, the overall length of the Staple nucleic acid may be 26 to 40 nucleotides in length, but is not limited to these lengths. The lengths of the regions where the Staple nucleic acid hybridizes to the target DNA, i.e., the length of the one end side (e.g., 3' side) region and the other end side (e.g., 5' side) region, may be the same or different and are not limited.

[0030] The hybridization reaction between the first-generation Staple nucleic acid designed as described above and the target DNA can be carried out either in vivo or in vitro. For example, · By mixing the Staple nucleic acid in a solution containing the target DNA and annealing it, · By simply mixing the Staple nucleic acid in a solution containing the target DNA, · By introducing a short hairpin expression vector and expressing the Staple nucleic acid from within the cell, It can be carried out. The hybridization reaction between the target DNA and the Staple nucleic acid can be carried out, for example, at a reaction temperature of 24 to 55°C for, for example, 1 minute to 2 days.

[0031] In addition, the ability of the first-generation Staple nucleic acid to induce the formation of a guanine quadruplex structure is realized by the presence of four guanine repeat sequences on the target DNA, the Staple nucleic acid hybridized to the target DNA changing the three-dimensional structure of the target DNA, the spatial distance between the four guanine repeat sequences on the target DNA becoming shorter, and as a result, inducing the guanine quadruplex structure by the four guanine repeat sequences on the target DNA.

[0032] The formation reaction of the guanine quadruplex structure using the first-generation Staple nucleic acid of the present invention can be carried out, for example, by causing a hybridization reaction between the target DNA and the Staple nucleic acid and then leaving it standing as it is. The formation reaction of the guanine quadruplex structure may be the same as the temperature and time for carrying out the hybridization reaction between the target DNA and the Staple nucleic acid, for example, at a reaction temperature of 24 to 55°C for, for example, 1 minute to 2 days. Confirmation of the formation of the guanine quadruplex structure can be carried out, for example, by in vitro translation assay, fluorescence signal measurement method by reacting with thioflavin T, stop assay method, etc.

[0033] An example of the specific structure of the first-generation Staple nucleic acid is shown in Chemical Formula 1 below. The oligonucleotide described as "short-chain nucleic acid" in this Chemical Formula 1 means the Staple nucleic acid. By introducing this oligonucleotide, the four guanine repeat sequences on the target DNA are brought close to each other to induce the formation of a guanine quadruplex structure.

[0034]

Chemical Formula

[0035] Structure of the second-generation Staple nucleic acid The second-generation Staple nucleic acid that can be used in the present invention refers to a Staple nucleic acid that supplies a part of four guanine repeat sequences constituting a guanine quadruplex structure with an oligonucleotide. That is, this second-generation Staple nucleic acid is structurally characterized in that it specifically contains a guanine repeat sequence constituting a guanine quadruplex structure within the Staple nucleic acid itself.

[0036] The second-generation Staple nucleic acid that can be used in the present invention is a G-supplying type oligonucleotide that hybridizes to nucleotide sequence portions containing one to three guanine repeat sequences on a target DNA at the nucleotide sequences near any two of the guanine repeat sequences (for example, the 5'-side or 3'-side), and contains three to one guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, wherein the G-supplying type oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance between a total of four guanine repeat sequences of the guanine repeat sequences on the target DNA and the guanine repeat sequences on the G-supplying type oligonucleotide, and forms a guanine quadruplex structure by these four guanine repeat sequences.

[0037] Here, when it is stated that it "contains one to three guanine repeat sequences on the target DNA," it means that there may be at least one or more guanine repeat sequences for forming a guanine quadruplex structure on the target DNA, and there may be four or more guanine repeat sequences on the target DNA. When there are four or more guanine repeat sequences on the target DNA, it means that a guanine quadruplex structure is formed by one to three of these guanine repeat sequences. Also, the "number of guanine repeat sequences on the target DNA" means the number of guanine repeat sequences on the target DNA that are used to form a guanine quadruplex structure using the second-generation Staple nucleic acid of the present invention. For example, even when there are four or more guanine repeat sequences on the target DNA, when a guanine quadruplex structure is formed by two of these guanine repeat sequences and two guanine repeat sequences supplied by a G-supplying oligonucleotide, the "number of guanine repeat sequences on the target DNA" is meant to be two.

[0038] Generally, the guanine quadruplex structure in cells is a stable nucleic acid higher-order structure constructed when four guanine repeat sequences of three or more bases are present in a closely spaced arrangement of less than seven bases. The inventors of the present invention have found that even when there are one to three guanine repeat sequences present at positions spaced seven or more bases apart on the target DNA, by using the first nucleotide sequence and the second nucleotide sequence of the present invention designed appropriately, and a G-supplying oligonucleotide containing three to one guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, the total of four guanine repeat sequences, namely one to three guanine repeat sequences present on these target DNAs and three to one guanine repeat sequences present on the G-supplying oligonucleotide, can be artificially brought into spatial proximity, and an artificial guanine quadruplex structure can be induced on the target DNA.

[0039] In the present invention, when referring to a guanine repeat sequence in a target DNA to which a second-generation Staple nucleic acid binds, it means a continuous sequence containing three or more guanines present on the target DNA or a continuous sequence containing three or more guanines present in the Staple nucleic acid. Specific guanine repeat sequences include, for example, GGG, GNGG (N can be any base and the number can be one or more), etc., which are guanine repeat sequences that can be elements forming a guanine quadruplex structure reported in papers.

[0040] For the second-generation Staple nucleic acid of the present invention to form a guanine quadruplex structure intracellularly, the oligonucleotide needs to have · the ability to recognize the sequence of the target DNA, and · depending on the number of guanine repeat sequences present on the target DNA (however, when there are four or more guanine repeat sequences on the target DNA, it means using one to three of those guanine repeat sequences), the ability to supply guanine repeat sequences so that the total number of guanine repeat sequences becomes four, · the ability to induce the formation of a guanine quadruplex structure simultaneously.

[0041] The ability of the second-generation Staple nucleic acid to recognize the sequence of the target DNA is realized by the oligonucleotide, which is the second-generation Staple nucleic acid, containing a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence near the guanine repeat sequence on the target DNA (for example, the 5'-side or 3'-side).

[0042] In the present invention, the sequences of the first nucleotide sequence and the second nucleotide sequence in the second-generation Staple nucleic acid are different for each target DNA sequence, and two sequence portions on the target DNA are selected so that the spatial distance of four guanine repeat sequences, namely, one to three guanine repeat sequences on the target DNA and three to one guanine repeat sequences on the second-generation Staple nucleic acid, can be shortened, and the first nucleotide sequence and the second nucleotide sequence can be determined so as to hybridize to these two sequence portions.

[0043] The first nucleotide sequence and the second nucleotide sequence of the second-generation Staple nucleic acid can be designed as appropriate. For example, the first nucleotide sequence and the second nucleotide sequence of the second-generation Staple nucleic acid may be · nucleotide sequence portions near the guanine repeat sequence closest to the 3'-end side (for example, on the 5'-side or 3'-side), · nucleotide sequence portions near the guanine repeat sequence closest to the 5'-end side (for example, on the 5'-side or 3'-side) that hybridize thereto.

[0044] For example, but not limited to, as an example of the second-generation Staple nucleic acid of the present invention, · a nucleotide sequence on the downstream side (3'-side) of one of the guanine repeat sequences of the target DNA (for example, a region of 13 to 20 bases starting from bases within 20 bases from the immediate downstream side (3'-side) (for example, the 1st to 20th bases on the downstream side of the guanine repeat sequence), or · a nucleotide sequence on the upstream side (5'-side) of one of the guanine repeat sequences of the target DNA (for example, a nucleotide sequence (for example, a region of 13 to 20 bases on the upstream side (5'-side)) starting from bases within 20 bases from the immediate upstream side (5'-side) (for example, the 1st to 20th bases on the downstream side of the guanine repeat sequence), etc. are selected, and the first nucleotide sequence and the second nucleotide sequence can be designed so as to hybridize to these regions.

[0045] The first nucleotide sequence and the second nucleotide sequence of the second-generation Staple nucleic acid of the present invention can be appropriately designed according to their applications. For example, the following characteristics (however, not limited thereto): · At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes to a nucleotide sequence portion near a guanine repeat sequence present before the stop codon on the target DNA; · At least one of the first nucleotide sequence or the second nucleotide sequence hybridizes to a nucleotide sequence portion near a guanine repeat sequence present in the 5' untranslated region on the target DNA; · Both the first nucleotide sequence and the second nucleotide sequence hybridize to a nucleotide sequence portion near a guanine repeat sequence present in the 5' untranslated region on the target DNA; · Both the first nucleotide sequence and the second nucleotide sequence hybridize to a nucleotide sequence portion near a guanine repeat sequence present in the 3' untranslated region. It can be appropriately designed to have such characteristics, but when used for other applications, it should be appropriately designed according to the application.

[0046] The distance of the nucleotide sequence region on the target DNA to which the first nucleotide sequence and the second nucleotide sequence hybridize from the guanine repeat sequence of the target DNA (the number of nucleotides sandwiched between the nucleotide at the proximal end closest to the guanine repeat sequence of the first nucleotide sequence or the second nucleotide sequence and the G of the guanine repeat sequence) can be appropriately determined by calculations related to thermodynamic stability, and the distance (number of nucleotides) is not particularly limited.

[0047] Therefore, as the starting point of the region where the Staple nucleic acid hybridizes, preferably, it is a base within 20 bases (for example, the 1st to 20th bases on the downstream side or upstream side) from the immediate downstream side (3' side) or immediate upstream side (5' side) of the guanine repeat sequence, more preferably, it is a base within 15 bases (for example, the 1st to 15th bases on the downstream side or upstream side) from the immediate downstream side (3' side) or immediate upstream side (5' side) of the guanine repeat sequence, more preferably, it is a base within 10 bases (for example, the 1st to 10th bases on the downstream side or upstream side) from the immediate downstream side (3' side) or immediate upstream side (5' side) of the guanine repeat sequence, more preferably, it is a base within 5 bases (for example, the 1st to 5th bases on the downstream side or upstream side) from the immediate downstream side (3' side) or immediate upstream side (5' side) of the guanine repeat sequence, and more preferably, it is possible to select a base within 3 bases (for example, the 1st to 3rd bases on the downstream side or upstream side) from the immediate downstream side (3' side) or immediate upstream side (5' side) of the guanine repeat sequence.

[0048] The second-generation Staple nucleic acid can be composed of DNA, RNA, modified nucleic acid, or a combination thereof. The second-generation Staple nucleic acid of the present invention is one in which the first nucleotide sequence and the second nucleotide sequence are directly or indirectly linked via a linker to one or more guanine repeat sequences at positions 3 to 1. In the present invention, a preferred nucleic acid form can be selected for each region of the first nucleotide sequence, the second nucleotide sequence, the one or more guanine repeat sequences at positions 3 to 1, and the linker that may optionally be included.

[0049] Here, in the second-generation Staple nucleic acid, when referring to the linker that connects the first nucleotide sequence and the second nucleotide sequence, the number of bases other than the first nucleotide sequence, the second nucleotide sequence, and the guanine repeat sequence that exist between the first nucleotide sequence and the second nucleotide sequence, between the first nucleotide sequence and / or the second nucleotide sequence and the guanine repeat sequence on the Staple nucleic acid, and between the guanine repeat sequences on the Staple nucleic acid, refers to the linker of any nucleic acid. It is necessary that the second-generation Staple nucleic acid can change the three-dimensional structure of the target DNA in terms of the binding of the first nucleotide sequence to the target DNA and the binding of the second nucleotide sequence to the target DNA, thereby shortening the spatial distance between a total of four guanine repeat sequences, namely, the guanine repeat sequence on the target DNA and the guanine repeat sequences on the Staple nucleic acid.

[0050] The second-generation Staple nucleic acid is one in which the first nucleotide sequence, the second nucleotide sequence, and one to three guanine repeat sequences are directly or indirectly connected via a linker. The overall length of the Staple nucleic acid varies depending on the nucleotide sequence of the target DNA. For example, it can be of any length designed based on criteria generally known in the art, such as the GC content, Tm value, etc., and can also be of a nucleotide length longer than that according to its design. As an example, the overall length of the Staple nucleic acid may be 26 to 40 nucleotides in length, but it is not limited to these lengths and can be of a nucleotide length longer or shorter than this. The lengths of the regions where the Staple nucleic acid hybridizes to the target DNA, namely, the length of the one end side (e.g., 3' side) region and the other end side (e.g., 5' side) region, may be the same or different and are not limited.

[0051] The hybridization reaction between the second-generation Staple nucleic acid designed as described above and the target DNA can be carried out either in vivo or in vitro, similar to the case of the first-generation Staple nucleic acid. For example, · By mixing the Staple nucleic acid in a solution containing the target DNA and annealing, · By simply mixing the Staple nucleic acid in a solution containing the target DNA, · By introducing a short hairpin expression vector and expressing the Staple nucleic acid intracellularly, it can be carried out. The hybridization reaction between the target DNA and the Staple nucleic acid may be, for example, at a reaction temperature of 24 to 55 °C and, for example, for 1 minute to 2 days.

[0052] In one aspect of the present invention, the second-generation Staple nucleic acid is characterized in that it has the ability to supply guanine repeat sequences so that the number of guanine repeat sequences is four in total according to the number of guanine repeat sequences present on the target DNA. That is, regardless of the number of guanine repeat sequences present on the target DNA, when forming a guanine quadruplex structure using any one of the guanine repeat sequences present on the target DNA, the Staple nucleic acid supplies three guanine repeat sequences, and when forming a guanine quadruplex structure using any two of the guanine repeat sequences present on the target DNA, the Staple nucleic acid supplies two guanine repeat sequences, and when forming a guanine quadruplex structure using any three of the guanine repeat sequences present on the target DNA, the Staple nucleic acid can supply one guanine repeat sequence.

[0053] Thus, since the guanine repeat sequences for forming the guanine quadruplex structure can be supplied by the second-generation Staple nucleic acid, the second-generation Staple nucleic acid can be rephrased as a G-supplying oligonucleotide or a G-supplying Staple nucleic acid in the sense of an oligonucleotide that supplies guanine repeat sequences.

[0054] In addition, the ability of the second-generation Staple nucleic acid to induce the formation of a guanine quadruplex structure is realized by the presence of a total of four guanine repeat sequences, which are the guanine repeat sequences present on the target DNA and the guanine repeat sequences present on the second-generation Staple nucleic acid, the Staple nucleic acid hybridized to the target DNA changing the three-dimensional structure of the target DNA, and the spatial distance of the total of four guanine repeat sequences on the target DNA and the second-generation Staple nucleic acid becoming shorter. As a result, a guanine quadruplex structure is induced by the total of four guanine repeat sequences on the target DNA and the second-generation Staple nucleic acid.

[0055] The reaction for forming a guanine quadruplex structure using the second-generation Staple nucleic acid of the present invention can be carried out, for example, by causing a hybridization reaction between the target DNA and the Staple nucleic acid and then leaving it standing as it is. The reaction for forming a guanine quadruplex structure may be the same as the temperature and time for the hybridization reaction between the target DNA and the Staple nucleic acid. For example, the reaction temperature may be 24 to 55°C, and the reaction time may be, for example, 1 minute to 2 days. Confirmation of the formation of the guanine quadruplex structure can be carried out, for example, by an in vitro translation assay, a fluorescence signal measurement method by reacting with thioflavin T, a stop assay method, or the like.

[0056] An example of the specific structure of the second-generation Staple nucleic acid is shown in FIG. 2 below. In FIG. 2, the case where three guanine repeat sequences are supplied by oligonucleotides is designated as Pattern A (G3 supply), the case where two guanine repeat sequences are supplied by oligonucleotides is designated as Pattern B (G2 supply), and the case where one guanine repeat sequence is supplied by oligonucleotides is designated as Pattern C (G1 supply). By introducing this oligonucleotide, the spatial distance of four guanine repeat sequences, which is the combination of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the G supply-type oligonucleotide, becomes shorter, and the formation of a guanine quadruplex structure by these four guanine repeat sequences is induced.

[0057]

Figure

[0058] Function of nucleic acid oligonucleotide In both the first-generation Staple nucleic acid and the second-generation Staple nucleic acid of the present invention, as a result of the formation of a guanine quadruplex structure, similar functions can be exhibited. That is, the functions exhibited by the first-generation Staple nucleic acid can also be exhibited by the second-generation Staple nucleic acid, and the functions exhibited by the second-generation Staple nucleic acid can also be exhibited by the first-generation Staple nucleic acid. This is because the functions of the Staple nucleic acid of the present invention are caused by the formation of a guanine quadruplex structure, and in both the first-generation Staple nucleic acid and the second-generation Staple nucleic acid, the properties of the formed guanine quadruplex structure are the same.

[0059] (1) Structural change of target DNA In the present invention, as a result of the action of the Staple nucleic acid, a loop portion is formed in a part of the target DNA due to the formation of a guanine quadruplex structure by four guanine repeat sequences, and the structure of the target DNA can be changed.

[0060] The first-generation Staple nucleic acid and the second-generation Staple nucleic acid have shown in the present invention that they can function with DNA as the target DNA.

[0061] Therefore, in the present invention, with respect to a nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising: wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of a total of four guanine repeat sequences of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, and forms a guanine quadruplex structure by the four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA and changing the structure of the target DNA can be provided.

[0062] When the structure of the target DNA is changed by the Staple nucleic acid, it is possible to inhibit or enhance a function that may change due to the change in the DNA structure. For example, when a guanine quadruplex structure is formed in the ORF region on the DNA to change the DNA structure, although not limited thereto, the elongation reaction of RNA polymerase is inhibited and the transcription reaction is suppressed. Alternatively, when a guanine quadruplex structure is formed near the promoter region to change the DNA structure, although not limited thereto, the DNA structure such as the enhancer region or the silencer region changes, and the activator or the repressor cannot bind, and as a result, transcription is regulated (suppressed or enhanced).

[0063] (2) Regulation of RNA transcription from target DNA In the present invention, a guanine quadruplex structure is formed on the target DNA, and as a result of the change in the structure of the target DNA, the transcription of RNA from the target DNA can be regulated. Here, when regulating the transcription of RNA from the target DNA, it includes both the case of suppressing transcription and the case of enhancing transcription.

[0064] The transcription from DNA to RNA is a reaction in which, within a cell, the base sequence of the template strand [antisense strand] of DNA is read to synthesize a functional RNA (for example, mRNA) having a complementary sequence, and it is a reaction caused by RNA polymerase. RNA polymerase binds to the target DNA, slides on the DNA, recognizes the promoter (formation of the initiation complex), starts transcription (formation of the elongation complex), synthesizes RNA through steps of elongation and termination of the transcription reaction.

[0065] In the present invention, a guanine quadruplex structure is formed on the target DNA using a Staple nucleic acid, and as a result of the change in the structure of the target DNA, RNA polymerase cannot bind to the target DNA, or the progression of RNA polymerase bound to the target DNA in the 3'→5' direction on the target DNA stops at the position of the guanine quadruplex structure, thereby suppressing the RNA synthesis reaction. In order to exhibit such a function, for example, by designing the sequence of the Staple nucleic acid of the present invention so that a guanine quadruplex structure is formed in the promoter region of the target DNA or in the downstream region on the 5' side of the promoter region on the target DNA, the above-described function can be exhibited.

[0066] Further, in the present invention, a guanine quadruplex structure is formed on the target DNA using a Staple nucleic acid, and as a result of the change in the structure of the target DNA, a repressor cannot bind to the target DNA, thereby enhancing the RNA synthesis reaction. In order to exhibit such a function, for example, by designing the sequence of the Staple nucleic acid of the present invention so that a guanine quadruplex structure is formed in the repressor binding region of the target DNA, the above-described function can be exhibited.

[0067] Therefore, in the present invention, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence portion on the target DNA containing one to four guanine repeat sequences at a nucleotide sequence on the 5'-side or 3'-side of the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising: a method for suppressing transcription of RNA from a target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of four guanine repeat sequences in total of the guanine repeat sequences on the target DNA and the guanine repeat sequences on the oligonucleotide, forms a guanine quadruplex structure by the four guanine repeat sequences, forms a loop portion in a part of the target DNA, and inhibits the binding of RNA polymerase to the target DNA or inhibits the function of RNA polymerase. can be provided.

[0068] (3) Regulation of protein expression In the present invention, a guanine quadruplex structure is formed on the target DNA, the structure of the target DNA is changed, and as a result, the transcription of RNA from the target DNA is suppressed or enhanced, so that the expression of the protein from the target DNA can be regulated. Here, the term "regulation" may refer to the case of suppressing protein expression and the case of enhancing expression.

[0069] As a specific example of a method for suppressing protein expression, for example, as a result of the formation of a guanine quadruplex structure in the target DNA, RNA polymerase cannot bind to the target DNA, or the progression of RNA polymerase bound to the target DNA in the 3'→5' direction stops at the position of the guanine quadruplex structure, thereby stopping the synthesis of RNA from the target DNA. As a result, since the protein translation reaction from the RNA is suppressed, the expression of the protein from the target DNA can be suppressed. In order to exhibit such a function, for example, by designing the sequence of the Staple nucleic acid of the present invention so that a guanine quadruplex structure is formed in the promoter region of the target DNA or the 5'-side region of the promoter region on the target DNA, the above-described function can be exhibited.

[0070] As a specific example of a method for enhancing protein expression, for example, as a result of the formation of a guanine quadruplex structure in the target DNA, a repressor cannot bind to the target DNA, thereby allowing the synthesis of RNA from the target DNA. As a result, since the protein translation reaction from the RNA is enhanced, the expression of the protein from the target DNA can be enhanced. In order to exhibit such a function, for example, by designing the sequence of the Staple nucleic acid of the present invention so that a guanine quadruplex structure is formed in the repressor binding region of the target DNA, the above-described function can be exhibited.

[0071] Therefore, in the present invention, An oligonucleotide comprising a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence on the 5'-side or 3'-side of the guanine repeat sequence with respect to a nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, A method for suppressing protein expression from a target DNA, comprising: changing the three-dimensional structure of the target DNA by hybridizing the oligonucleotide to the target DNA of the first nucleotide sequence and the second nucleotide sequence, shortening the spatial distance of four guanine repeat sequences in total of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forming a guanine quadruplex structure by these four guanine repeat sequences, forming a loop portion in a part of the target DNA, inhibiting the binding of RNA polymerase to the target DNA or inhibiting the function of RNA polymerase, and suppressing the transcription of RNA from the target DNA can be provided.

[0072] (4) Replication inhibition of target DNA In the present invention, a guanine quadruplex structure is formed on the target DNA, and as a result of the change in the structure of the target DNA, the replication of the target DNA can be suppressed.

[0073] DNA replication occurs in cells by unwinding the double-stranded template DNA and replicating in different manners on the leading strand and the lagging strand. In the leading strand, which is one of the double strands, an RNA primer is synthesized at the origin of replication, and DNA polymerase bound to the template DNA moves along the template DNA in the 3'→5' direction while binding bases to the 3' end of the RNA primer, and replication is carried out by synthesizing the synthesized DNA in the 5'→3' direction. In the lagging strand, which is the other of the double strands, an RNA primer is synthesized, and DNA polymerase bound to the template DNA moves along the template DNA in the 3'→5' direction while binding bases to the 3' end of the RNA primer, synthesizing the synthesized DNA (Okazaki fragment) in the 5'→3' direction, and replicating by binding the synthesized DNA (Okazaki fragment).

[0074] In the present invention, a guanine quadruplex structure is formed on target DNA using a Staple nucleic acid, and as a result of the change in the structure of the target DNA, DNA polymerase cannot bind to the target DNA (for example, template DNA), or the progression of DNA polymerase bound to the target DNA (for example, template DNA) in the 3'→5' direction on the target DNA stops at the position of the guanine quadruplex structure, whereby the DNA synthesis reaction can be suppressed. In this case, when a guanine quadruplex structure is formed in the leading strand, replication from the leading strand stops, and when a guanine quadruplex structure is formed in the lagging strand, replication from the lagging strand stops. Since replication stops regardless of the region on the target DNA where the guanine quadruplex structure is formed, by designing the sequence of the Staple nucleic acid of the present invention such that a guanine quadruplex structure is formed in any region on the target DNA, such a replication suppression function can be exhibited.

[0075] Therefore, in the present invention, An oligonucleotide comprising a nucleotide sequence portion containing one to four guanine repeat sequences on target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridization of the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of four guanine repeat sequences including the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, and forms a guanine quadruplex structure by the four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA and suppressing the replication of the target DNA by inhibiting the binding of DNA polymerase to the target DNA or inhibiting the function of DNA polymerase can be provided.

[0076] Use of the Staple nucleic acid of the present invention Since the Staple nucleic acids (oligonucleotides) of the present invention have the functions as described above in the section of "Functions of oligonucleotides", by using these functions, the present invention can provide a pharmaceutical composition containing the Staple nucleic acids of the present invention.

[0077] That is, when using the function of regulating the transcription of target DNA or the function of regulating the expression of proteins as the function of the Staple nucleic acids of the present invention, diseases caused by abnormal transcription from target DNA to RNA, such as cancer, autoimmune diseases, inflammatory diseases, neuropsychiatric diseases, diabetes, cardiovascular diseases and other diseases (Cell. 2013 March 14; 152(6): 1237-1251), or diseases caused by abnormal expression of proteins from target DNA, such as cancer, neuropsychiatric diseases and other diseases (Journal of Pathology, J Pathol 2010; 220: 140-151), a pharmaceutical composition for treating these diseases can be provided. Further, when using the function of suppressing the replication of target DNA as the function of the Staple nucleic acids of the present invention, a pharmaceutical composition for treating diseases caused by abnormal replication of target DNA, such as cancer, viral diseases and other diseases (Melvin L. DePamphilis, "DNA replication and human disease", Cold Spring Harbor Laboratory Press c2006) can be provided.

[0078] The pharmaceutical composition containing the Staple nucleic acid of the present invention may be in the form of administering the Staple nucleic acid prepared in vitro, or in the form of administering a vector that produces the Staple nucleic acid to a living body to produce the Staple nucleic acid in vivo. Therefore, the pharmaceutical composition of the present invention can be prepared by formulating a therapeutically effective amount of the Staple nucleic acid of the present invention alone or together with a pharmaceutically acceptable carrier (additive), excipient, and / or diluent, or a vector capable of producing a therapeutically effective amount of the Staple nucleic acid of the present invention in vivo can be prepared by formulating it alone or together with a pharmaceutically acceptable carrier (additive), excipient, and / or diluent.

[0079] Here, the term "therapeutically effective amount" as used herein refers to the amount of the Staple nucleic acid of the present invention that is effective for obtaining a therapeutic effect based on the desired main effect, regardless of the presence or absence of side reactions.

[0080] The "pharmaceutically acceptable carrier" used herein refers to those generally used in the art and means a carrier suitable for application to human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications. Such carriers include, but are not limited to, pharmaceutically acceptable materials, compositions, or vehicles involved in the transport or delivery of the compound of interest from one organ or part of the body to another, such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium, calcium stearate, zinc stearate, or stearic acid), or solvents containing the material.

[0081] The pharmaceutical composition containing the Staple nucleic acid of the present invention can be used in combination with other components. There is no particular limitation on such other components, and it may be used in combination with any kind of component.

[0082] Since the Staple nucleic acid (oligonucleotide) of the present invention also has the functions as described above in the section of "Functions of oligonucleotides", kits for realizing these functions containing the Staple nucleic acid of the present invention can be provided. That is, · A kit for structural change of target DNA containing a Staple nucleic acid for realizing the function of "(1) Structural change of target DNA" described above, · A kit for transcriptional regulation of RNA from target DNA containing a Staple nucleic acid for realizing the function of "(2) Transcriptional regulation of RNA from target DNA" described above, · A kit for protein expression regulation containing a Staple nucleic acid for realizing the function of "(3) Protein expression regulation" described above, · A kit for replication inhibition of two types of target DNA containing a Staple nucleic acid for realizing the function of "(4) Replication inhibition of target DNA" described above, can be provided. However, as the term of the Staple nucleic acid of the present invention expands, the content of the kit can also be expanded.

[0083] For example, as a more specific embodiment, for example, as a kit for transcriptional regulation of RNA from target DNA containing a Staple nucleic acid for realizing the function of "(2) Transcriptional regulation of RNA from target DNA" described above, a first nucleotide sequence and a second nucleotide sequence that hybridize to the nucleotide sequence on the 5' side or 3' side of the guanine repeat sequence with respect to a nucleotide sequence portion containing one to four guanine repeat sequences on the target DNA, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, an oligonucleotide comprising, When the oligonucleotide hybridizes to the target DNA of the first nucleotide sequence and the second nucleotide sequence, it changes the three-dimensional structure of the target DNA, shortening the spatial distance between four guanine repeat sequences, namely, the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, and forming a guanine quadruplex structure by these four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA and inhibiting the binding of RNA polymerase to the target DNA or inhibiting the function of RNA polymerase, and regulating the transcription of RNA from the target DNA, said oligonucleotide A protein expression regulation kit containing can be provided.

[0084] Hereinafter, the present invention will be specifically illustrated with reference to examples. The examples shown below do not limit the present invention in any way.

Examples

[0085] Example 1: Confirmation of induction of DNA guanine quadruplex structure formation by the first-generation Staple nucleic acid In this example, it was confirmed that the first-generation Staple nucleic acid targeting DNA induces the formation of a DNA guanine quadruplex structure in the target DNA.

[0086] The first-generation Staple nucleic acid targeting the 1+3 type 158 nt Template DNA sequence In this experiment, a 158-base model nucleic acid DNA sequence (1+3 type 158 nt Template DNA) (SEQ ID NO: 1) having four guanine repeat sequences was designed and used. The sequence of the 1+3 type 158 nt Template DNA has four guanine repeat sequences in the target DNA, and a guanine quadruplex structure can be formed by the binding of the first-generation Staple nucleic acid of the present invention.

[0087] As a comparison, Mutant 158 nt Template DNA (SEQ ID NO: 2) was created by mutating all four guanine repeat sequences present on the 1+3 type 158 nt Template DNA, which is the target DNA, to adenine repeat sequences. This is a comparison control where even if the first-generation Staple nucleic acid of the present invention binds, a quadruplex structure cannot be formed.

[0088] The base sequences of the 1+3 type 158 nt Template DNA used as the target DNA and the Mutant 158 nt Template DNA are as follows.

[0089]

Chemical formula

[0090] For this target DNA, an oligonucleotide (Staple nucleic acid) with a length of 50 mer was designed and prepared. That is, in designing this oligonucleotide, based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target DNA and the guanine repeat sequences on the target DNA, as well as the lengths of the first nucleotide sequence and the second nucleotide sequence, a 50 mer oligonucleotide was designed. The prepared oligonucleotide arranges a 25-base first nucleotide sequence and a 25-base second nucleotide sequence starting from the second base from two guanine repeat sequences on the 3'-terminal side.

[0091]

Chemical formula

[0092] All the oligonucleotides used in this specification were purchased from Thermo Fisher Scientific, Inc. Also, all samples were used after desalting and purification.

[0093] When adding the Staple nucleic acid, 1.8 μl of each of the above Template sequences at 100 μM, 2.2 μL of the 100 μM above-mentioned Staple nucleic acid, and 6.0 μl of 10× annealing Buffer (1 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and 40.0 μL of milliQ was added to this solution, followed by annealing by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. To this solution, 10 μL of ThT (3 μM), a probe that emits a fluorescent signal by binding to the guanine quadruplex structure, was added and incubated for 30 minutes.

[0094] On the other hand, when not adding the Staple nucleic acid, 1.8 μl of each of the above Template sequences at 100 μM and 6.0 μl of 10× annealing Buffer (1 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and 42.2 μL of milliQ was added to this solution, followed by annealing by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. 10 μL of 3 μM ThT was added to this solution and incubated for 30 minutes.

[0095] The formation of the guanine quadruplex structure was confirmed by measuring the fluorescent signal from the probe ThT, which emits a fluorescent signal by binding to the guanine quadruplex structure. Fluorescence measurement was performed using an Agilent Cary Eclipse spectrophotometer under the conditions of excitation wavelength: 440 nm, measurement wavelength: 450 - 600 nm, sweep speed: 120 nm / min, and PMT Voltage: 850 V.

[0096] The results are shown in Figure 1. In this Figure 1, · Those in which the Staple nucleic acid was reacted with the 1 + 3 type 158 nt Template DNA, · Those in which the Staple nucleic acid was not added to the 1 + 3 type 158 nt Template DNA, · Those in which the Staple nucleic acid was reacted with the Mutant 158 nt Template DNA, · Those without addition of Staple nucleic acid to Mutant 158 nt Template DNA The measurement results for the following 4 conditions are shown.

[0097] When Staple nucleic acid was added to 1 + 3 Template DNA, a significant increase in the fluorescence signal was confirmed. On the other hand, in the case of Mutant 158 nt Template DNA in which guanine, which is a component of the guanine quadruplex structure in the 1 + 3 type 158 nt Template, was converted to adenine, no significant change in the fluorescence signal was confirmed regardless of the presence or absence of Staple nucleic acid. These results indicate that, as originally assumed, the addition of Staple nucleic acid can induce the formation of DNA guanine quadruplex structure.

[0098] Example 2: Confirmation of induction of DNA guanine quadruplex structure formation by the second-generation Staple nucleic acid In this example, it was confirmed that the second-generation type Staple nucleic acid targeting DNA induces the formation of DNA guanine quadruplex structure in the target DNA.

[0099] The second-generation type Staple nucleic acid targeting the 1 + 1 type 160 nt Template DNA sequence In this experiment, a 160-base model nucleic acid DNA sequence (1 + 1 type 160 nt Template DNA) (SEQ ID NO: 6) having two guanine repeat sequences was designed and used. The sequence of the 1 + 1 type 160 nt Template DNA has two guanine repeat sequences in the target DNA, and by binding of the second-generation type Staple nucleic acid of the present invention containing two guanine repeat sequences, two guanine repeat sequences derived from the 1 + 1 type 160 nt Template DNA and two guanine repeat sequences derived from the second-generation type Staple nucleic acid can form a guanine quadruplex structure.

[0100] As a comparative control, Mutant 160 nt Template DNA (SEQ ID NO: 7) was created by mutating all two guanine repeat sequences present on the 1+1 type 160 nt Template DNA, which is the target DNA, into adenine repeat sequences. This is a comparative control where a quadruplex structure cannot be formed even when the second-generation Staple nucleic acid of the present invention binds.

[0101] The base sequences of the 1+1 type 160 nt Template DNA as the target DNA and the Mutant 160 nt Template DNA are as follows.

[0102]

Chemical formula

[0103] For this target DNA, an oligonucleotide (Staple nucleic acid) with a length of 50 mer was designed and prepared. That is, in the design of this oligonucleotide, based on the positional relationship between the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target DNA and the guanine repeat sequences on the target DNA, and the positional relationship between the two guanine repeat sequences on the Staple nucleic acid, and the lengths of the first nucleotide sequence and the second nucleotide sequence, a 50 mer oligonucleotide was designed. The prepared oligonucleotide arranges a 25-base first nucleotide sequence on the 3'-side starting from the second base from the most 3'-side guanine of the guanine repeat sequence on the 5'-end side, and a 25-base second nucleotide sequence on the 5'-side starting from the second base from the most 5'-side guanine of the guanine repeat sequence on the 3'-end side.

[0104]

Chemical formula

[0105] All the oligonucleotides used in this specification were purchased from Thermo Fisher Scientific, Inc. Also, all samples were used after desalting and purification.

[0106] When adding the Staple nucleic acid, 1.8 μl of each of the above Template sequences at 100 μM, 2.2 μL of the above Staple nucleic acid at 100 μM, and 6.0 μl of 10× annealing Buffer (1 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and 40.0 μL of milliQ was added to this solution, followed by annealing by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. To this solution, 10 μL of ThT (3 μM), which is a probe that emits a fluorescence signal by binding to the guanine quadruplex structure, was added and incubated for 30 minutes.

[0107] On the other hand, when not adding the Staple nucleic acid, 1.8 μl of each of the above Template sequences at 100 μM and 6.0 μl of 10× annealing Buffer (1 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and 42.2 μL of milliQ was added to this solution, followed by annealing by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. To this solution, 10 μL of 3 μM ThT was added and incubated for 30 minutes.

[0108] The formation of the guanine quadruplex structure was confirmed by measuring the fluorescence signal from the probe ThT, which emits a fluorescence signal by binding to the guanine quadruplex structure. Fluorescence measurement was performed using an Agilent Cary Eclipse spectrophotometer under the conditions of excitation wavelength: 440 nm, measurement wavelength: 450 - 600 nm, sweep speed: 120 nm / min, and PMT Voltage: 850 V.

[0109] The results are shown in Figure 2. The upper figure in Figure 2 shows the results of the experiment using 1 + 1 type 160 nt Template DNA, and the lower figure in Figure 2 shows the results of the experiment using Mutant 160 nt Template DNA. In the upper figure of Figure 2, · Those obtained by reacting staple nucleic acids with 1 + 1 type 160 nt template DNA, · Those without adding staple nucleic acids to 1 + 1 type 160 nt template DNA, · Those obtained by reacting A - supplied type staple nucleic acids with 1 + 1 type 160 nt template DNA, show the measurement results for these 3 conditions. In the lower figure of Figure 2, · Those obtained by reacting staple nucleic acids with Mutant 160 nt template DNA, · Those without adding staple nucleic acids to Mutant 160 nt template DNA, · Those obtained by reacting A - supplied type staple nucleic acids with Mutant 160 nt template DNA, show the measurement results for these 3 conditions.

[0110] When staple nucleic acids were added to 1 + 1 type 160 nt template DNA, a significant increase in the fluorescence signal was confirmed. Also, even when adding A - supplied type staple nucleic acids that supply adenine repeat sequences instead of guanine repeat sequences, no increase in the fluorescence signal was observed. On the other hand, in the case of Mutant 160 nt template DNA in which guanine, which is a component of the guanine quadruplex structure in 1 + 1 type 160 nt template DNA, was converted to adenine, no significant change in the fluorescence signal was confirmed regardless of the presence or absence of the second - generation type staple nucleic acids, or when A - supplied type staple nucleic acids were added (lower figure of Figure 2). In this lower figure of Figure 2, the graphs of "Mutant 160 nt template DNA, without staple nucleic acids" and "Mutant 160 nt template DNA, with A - supplied type staple nucleic acids" completely overlap. These results indicate that, as initially assumed, the addition of the second - generation type staple nucleic acids can induce the formation of the DNA guanine quadruplex structure.

[0111] Example 3: Confirmation of transcription suppression effect by the first-generation Staple nucleic acid In this example, the transcription suppression effect of the first-generation Staple nucleic acid targeting DNA was confirmed from the target DNA.

[0112] First-generation Staple nucleic acid targeting 1+3 type 158 nt Template DNA In this example, the 158-base model nucleic acid DNA sequence (1+3 type 158 nt Template DNA) having four guanine repeat sequences prepared in Example 1 was used.

[0113] For this target DNA, in addition to the 50 mer Staple nucleic acid prepared in Example 1, the following two types of oligonucleotides (Staple nucleic acids) with different lengths were designed and prepared. That is, in the design of these oligonucleotides, from the positional relationship of the guanine repeat sequences on the target DNA at the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target DNA, and the lengths of the first nucleotide sequence and the second nucleotide sequence, a 50 mer oligonucleotide was used as the Staple nucleic acid of SEQ ID NO: 3, a 40 mer oligonucleotide shortened by 10 mer from the Staple nucleic acid of SEQ ID NO: 3 (placing the first nucleotide sequence and the second nucleotide sequence of 20 bases each from the second base of the two guanine repeat sequences, SEQ ID NO: 4), and a 30 mer oligonucleotide shortened by 20 mer from the Staple nucleic acid of SEQ ID NO: 3 (placing the first nucleotide sequence and the second nucleotide sequence of 15 bases each from the second base of the two guanine repeat sequences, SEQ ID NO: 5) were designed and created. The sequences of each Staple nucleic acid are as follows.

[0114]

Chemical formula

[0115] All the oligonucleotides used in this specification were purchased from Thermo Fisher Scientific, Inc. Also, all samples were used after desalting and purification.

[0116] Using these oligonucleotides (Staple nucleic acids), the inhibitory function of transcription as a result of the construction of a guanine quadruplex structure by the first-generation Staple nucleic acids against target DNA was evaluated by electrophoresis of the transcription products.

[0117] 3.6 μl of 100 μM of the above template sequence (1+3 type 158 nt Template DNA) and 3.6 μl of 100 μM T7 promoter complementary strand complementary to the above T7 promoter sequence, 9.0 μl of 10× annealing buffer (1.5 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and this was designated as Premix A.

[0118] To 3.6 μl of this Premix A, 1.6 μl of 100 μM Staple nucleic acid (or 1.6 μl of water in the case of the control without Staple nucleic acid) and 2.8 μl of water were added, and annealing was performed by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. This solution was designated as Premix B.

[0119] To 8 μl of Premix B, 10× transcription buffer (500 mM NaCl, 80 mM MgCl 2 2, 50 mM DTT, 400 mM Tris-HCl (pH 8.0)), 2 μl of each NTP, and 2 μl of T7 RNA polymerase were added, and a transcription reaction was carried out under the condition of 37 °C for 30 minutes. Thereafter, DNase treatment etc. were performed, electrophoresis using denaturing PAGE (12%) was carried out, and then staining and evaluation were performed.

[0120] The results are shown in Fig. 3. When no Staple nucleic acid was added (the lane of (2) (w / o Staple nucleic acid) in Fig. 3), RNA polymerase could transcribe to the end of the target DNA, and an RNA product of about 131 mer could be obtained. On the other hand, when the Staple nucleic acid was introduced to induce the formation of a guanine quadruplex structure on the target DNA, a band could be confirmed at the 39 mer position. As shown in this result, successful transcription suppression was confirmed in any sample with different lengths of the Staple nucleic acid. Note that the activity of transcription suppression was not affected by the differences in the lengths of the three types of Staple nucleic acids.

[0121] Example 4: Confirmation of transcription suppression effect by the second-generation Staple nucleic acid In this example, the transcription suppression effect of the second-generation Staple nucleic acid targeting DNA was confirmed.

[0122] Second-generation Staple nucleic acid targeting 1+1 type 160 nt Template DNA In this example, the 160-base model nucleic acid DNA sequence (1+1 type 160 nt Template DNA) with two guanine repeat sequences prepared in Example 2 was used.

[0123] In addition to the 71-mer G-supplying Staple nucleic acid prepared in Example 2, the following two types of oligonucleotides (Staple nucleic acids) with different lengths and a 71-mer A-supplying Staple nucleic acid in which the guanine repeat sequence was replaced with an adenine repeat sequence were prepared for this target DNA. That is, in designing these oligonucleotides, based on the binding sites of the first nucleotide sequence and the second nucleotide sequence on the target DNA and the positional relationship of the guanine repeat sequence on the target DNA, a 71-mer oligonucleotide was used as the Staple nucleic acid of SEQ ID NO: 8, a 61-mer oligonucleotide shortened by 10 mer from the Staple nucleic acid of SEQ ID NO: 8 (the first nucleotide sequence and the second nucleotide sequence of 20 bases each from the second base of the two guanine repeat sequences are arranged so as to sandwich the sequence supplying the two 11-base guanine repeat sequences, SEQ ID NO: 9), and a 51-mer oligonucleotide shortened by 20 mer from the Staple nucleic acid of SEQ ID NO: 8 (the first nucleotide sequence and the second nucleotide sequence of 15 bases each from the second base of the two guanine repeat sequences are arranged so as to sandwich the sequence supplying the two 11-base guanine repeat sequences, SEQ ID NO: 10) were designed and prepared. Note that the A-supplying Staple nucleic acid (SEQ ID NO: 11) has the nucleotide sequence unchanged except that the guanine repeat sequence in the G-supplying Staple nucleic acid of SEQ ID NO: 8 was replaced with an adenine repeat sequence.

[0124]

Chemical formula

[0125] All the oligonucleotides used in this specification were purchased from Thermo Fisher Scientific, Inc. Also, all samples were used after desalting and purification.

[0126] Using these oligonucleotides (Staple nucleic acids), the suppression function of transcription as a result of the construction of a guanine quadruplex structure by the second-generation Staple nucleic acid against the target DNA was evaluated by electrophoresis of the transcription product.

[0127] 3.6 μl of 100 μM of the above template sequence (1+1 type 160 nt Template DNA) and 3.6 μl of 100 μM T7 promoter complementary strand complementary to the above T7 promoter sequence, 9.0 μl of 10× annealing buffer (1.5 M KCl, 500 mM Tris-HCl (pH 7.6)) were mixed, and this was designated as Premix A.

[0128] To 3.6 μl of this Premix A, 1.6 μl of 100 μM Staple nucleic acid (in the case of the control without Staple nucleic acid, 1.6 μl of water) and 2.8 μl of water were added, and annealing was performed by lowering the temperature from 90 °C to 20 °C at a rate of 1 °C per minute. This solution was designated as Premix B.

[0129] To 8 μl of Premix B, 10× transcription buffer (500 mM NaCl, 80 mM MgCl 2 , 50 mM DTT, 400 mM Tris-HCl (pH 8.0)), 2 μl of each NTP, and 2 μl of T7 RNA polymerase were added, and a transcription reaction was carried out under the condition of 37 °C for 30 minutes. Thereafter, DNase treatment etc. was performed, electrophoresis using denaturing PAGE (12%) was carried out, and then staining and evaluation were performed.

[0130] The results are shown in Figure 4. When no Staple nucleic acid was added (lane (2) in Figure 4, "w / o Staple nucleic acid"), when the template was Mutant 160 nt Template DNA (lane (6) in Figure 4, "71 mer Staple nucleic acid (Mutant Template)"), and when the A-supplied Staple nucleic acid was added (lane (7) in Figure 4, "71 mer Staple nucleic acid (A-supplied Staple nucleic acid)"), RNA polymerase was able to transcribe to the end of the target DNA, and a 133 mer RNA product could be obtained. On the other hand, when a G-supplied Staple nucleic acid was introduced to induce the formation of a guanine quadruplex structure on the target DNA, a band could be confirmed at the 39 mer position. As shown in these results, successful transcription suppression was confirmed in any sample in which the length of the G-supplied Staple nucleic acid was changed. The activity of transcription suppression was not affected by the differences in the lengths of the three types of G-supplied Staple nucleic acids.

Industrial Applicability

[0131] The present invention shows that the previously developed Staple nucleic acid can form a guanine quadruplex structure on the target DNA by a total of four guanine repeat sequences including the guanine repeat sequence on the target DNA, and can change the structure of the target DNA. As a result of such an action, a novel use of the Staple nucleic acid (such as regulation of transcription from DNA to RNA) can be provided.

Sequence Listing Free-Text

[0132] ·SEQ ID NO: 1: 1 + 3 type 158 nt Template DNA ·SEQ ID NO: 2: Mutant 158 nt Template DNA ·SEQ ID NO: 3: First-generation Staple nucleic acid (50 mer) ·SEQ ID NO: 4: First-generation Staple nucleic acid (40 mer) ·SEQ ID NO: 5: First-generation Staple nucleic acid (30 mer) ·SEQ ID NO: 6: 1+1 type 160 nt Template DNA ·SEQ ID NO: 7: Mutant 160 nt Template DNA ·SEQ ID NO: 8: Second-generation Staple nucleic acid (71 mer) ·SEQ ID NO: 9: Second-generation Staple nucleic acid (61 mer) ·SEQ ID NO: 10: Second-generation Staple nucleic acid (51 mer) ·SEQ ID NO: 11: A-supplying Staple nucleic acid (71 mer)

Claims

1. An oligonucleotide comprising a nucleotide sequence portion containing one to four guanine repeat sequences on a target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence near the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridizing the first nucleotide sequence and the second nucleotide sequence to the target DNA, shortens the spatial distance of a total of four guanine repeat sequences of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, and forms a guanine quadruplex structure by the four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA and changing the structure of the target DNA.

2. The method for changing the structure of a target DNA according to claim 1, wherein the first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are located near the guanine repeat sequence on the target DNA - a nucleotide sequence portion on the 5'- or 3'-side of the guanine repeat sequence on the 3'-terminal side, - a nucleotide sequence portion on the 5'- or 3'-side of the guanine repeat sequence on the 5'-terminal side and hybridize thereto.

3. The method for changing the structure of a target DNA according to claim 1 or 2, wherein the oligonucleotide is composed of DNA, RNA, modified nucleic acid, or a combination thereof.

4. An oligonucleotide comprising a nucleotide sequence portion containing one to four guanine repeat sequences on a target DNA, a first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence near the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, wherein A method for regulating RNA transcription from a target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridizing to the target DNA of the first nucleotide sequence and the second nucleotide sequence, shortens the spatial distance of four guanine repeat sequences in total of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forms a guanine quadruplex structure by the four guanine repeat sequences, forms a loop portion in a part of the target DNA, and inhibits the binding of RNA polymerase to the target DNA or inhibits the function of RNA polymerase. **Claim 5** The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA - The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 3' end side, - The nucleotide sequence portion on the 5' side or 3' side of the guanine repeat sequence on the 5' end side The method for regulating RNA transcription from a target DNA according to claim 4, which hybridizes thereto. **Claim 6** The method for regulating RNA transcription from a target DNA according to claim 4 or 5, wherein the oligonucleotide is composed of DNA, RNA, a modified nucleic acid, or a combination thereof. **Claim 7** An oligonucleotide comprising a first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence portion containing one to four guanine repeat sequences on a target DNA at a nucleotide sequence in the vicinity of the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, A method for regulating protein expression from a target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridizing to the target DNA of the first nucleotide sequence and the second nucleotide sequence, shortens the spatial distance of four guanine repeat sequences in total of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forms a guanine quadruplex structure by these four guanine repeat sequences, forms a loop portion in a part of the target DNA, inhibits the binding of RNA polymerase to the target DNA or inhibits the function of RNA polymerase, and regulates the transcription of RNA from the target DNA.

8. The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA - The nucleotide sequence part on the 5' side or 3' side of the guanine repeat sequence on the 3' end side, - The nucleotide sequence part on the 5' side or 3' side of the guanine repeat sequence on the 5' end side The method for regulating protein expression from a target DNA according to claim 7, which hybridizes to the above.

9. The method for regulating protein expression from a target DNA according to claim 7 or 8, wherein the oligonucleotide is DNA, RNA, a modified nucleic acid, or a combination thereof.

10. An oligonucleotide comprising a first nucleotide sequence and a second nucleotide sequence that hybridize to a nucleotide sequence portion containing one to four guanine repeat sequences on a target DNA at a nucleotide sequence near the guanine repeat sequence, and three to zero guanine repeat sequences according to the number of guanine repeat sequences on the target DNA, A method for suppressing replication of target DNA, wherein the oligonucleotide changes the three-dimensional structure of the target DNA by hybridizing to the target DNA of the first nucleotide sequence and the second nucleotide sequence, shortens the spatial distance of four guanine repeat sequences in total of the guanine repeat sequence on the target DNA and the guanine repeat sequence on the oligonucleotide, forms a guanine quadruplex structure by the four guanine repeat sequences, thereby forming a loop portion in a part of the target DNA, and inhibiting the binding of DNA polymerase to the target DNA or inhibiting the function of DNA polymerase.

11. The first nucleotide sequence and the second nucleotide sequence of the oligonucleotide are in the vicinity of the guanine repeat sequence on the target DNA - the nucleotide sequence portion on the 5' side or the 3' side of the guanine repeat sequence on the 3' end side, - the nucleotide sequence portion on the 5' side or the 3' side of the guanine repeat sequence on the 5' end side The method for suppressing replication of target DNA according to claim 10, which hybridizes to.

12. The method for suppressing replication of target DNA according to claim 10 or 11, wherein the oligonucleotide is composed of DNA, RNA, modified nucleic acid, or a combination thereof.

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