WO2011106997A1

WO2011106997A1 - Novel 10-23 deoxyribozyme analogues and uses thereof

Info

Publication number: WO2011106997A1
Application number: PCT/CN2011/000497
Authority: WO
Inventors: 刘克良; 何军林; 张迪; 王�琦; 徐亮
Original assignee: 中国人民解放军军事医学科学院毒物药物研究所
Priority date: 2010-03-04
Filing date: 2011-03-24
Publication date: 2011-09-09
Also published as: CN102191233A; CN102191233B

Abstract

Novel 10-23 deoxyribozyme analogues and uses thereof are provided. The method for preparing the 10-23 deoxyribozyme analogues, the medicine kit, kit or composition containing the 10-23 deoxyribozyme analogues and the uses thereof are also provided. Based on the different conservatism of each nucleotide acid unit in catalytic domain of 10-23 deoxyribozyme and the different contributions of the functional groups of bases to the catalytic reaction, purine bases and pyrimidine bases in catalytic domain are substituted by their analogues, and the novel 10-23 deoxyribozyme analogues with higher catalytic efficiency are obtained.

Description

Novel 10-23 deoxyribozyme analogs and uses thereof

The present invention relates to a novel class of deoxyribozyme analogs, in particular to a novel class

10-23 denuclease analogs, and their use. Background technique

Ribozymes are a class of natural nucleic acid molecules that catalyze the cleavage of nucleic acids. They use their advanced structures and metal ions, water molecules, etc. in their environment to achieve the function of catalytically cleavage of nucleic acids. The discovery of ribozymes not only gives us a new understanding of the function of nucleic acids, but also provides a new tool for the research and application of nucleic acids. Through the efforts of scientists, ribozymes have been applied to the study of catalytically cleavage of disease-causing genes and inactivation. Therefore, ribozymes have become another class of gene therapy candidates following antisense drugs. To date, a variety of ribozyme-based drugs are in the research and development stage. However, the in vivo application of ribozymes is limited by the following factors, their chemical instability and enzyme instability, and the ribozyme transport to the target to overcome obstacles.

The discovery of deoxyribozymes provides an alternative to improving the pharmaceutical properties of ribozymes, since the chemical and enzymatic stability of deoxyribozymes is much higher and synthesis is simpler. The most promising deoxyribozyme is the 10-23 deoxyribozyme, which has been studied to date for oncogenes, viruses, and genetic mutations. Its clinical application is limited by its inability to express in vivo. There are also difficulties in the delivery of therapeutic drugs. However, with the development of gene therapy and transport technology, the transport of exogenous deoxyribozymes will be solved. Another drawback is that its effectiveness requires the participation of divalent metal ions (such as M _g ²⁺ ) that are much higher than physiological concentrations. Within a certain range, the higher the concentration of M _g ^{2+ , the} higher the catalytic efficiency. Under simulated physiological conditions, 2 mM MgCl ₂ , 150 mM KCl, pH 7.5, 37. C, ^=0.01 mm ¹ . While the physiological concentration of Mg ^{2+ in} cells is only 0.1-0.2 mM, the catalytic efficiency of cystoxyzyme is far below the requirement for therapeutic purposes. So looking for low Mg ²⁺ The new deoxyribozyme with high catalytic activity at the concentration will be one of the key breakthrough points of the nucleus as a gene therapy drug.

A schematic diagram of the binding of 10-23 degassed ribozyme to the substrate is shown in Figure 1. In the figure, N is the nucleotide monomer composition of the substrate, and Ν is the nucleotide sequence of the 10-23 deoxyribozyme recognition domain. The body composition, Ν is any nucleotide unit with a target nucleotide Ν, which can perform WC complementary pairing; R is a purine nucleotide unit, Υ is a pyrimidine nucleotide unit, and the length of the mutual moiety can follow the target nucleic acid Changes in sequence, composition, etc., the number of 3⁄4 bases at both ends of the deoxyribozyme is n (for example, n=4-50

Based on the great potential of gene therapy drugs, the chemical modification of it is to optimize its pharmaceutical properties, such as improving its enzyme stability and transport efficiency. On the other hand, chemical modification is used to explore its catalytic mechanism and how to further improve its catalytic efficiency. So far, the mechanism and catalytic efficiency of this deoxyribozyme have been reported by chemical modification, but no breakthrough has been made in both the mechanism and the catalytic efficiency. The chemical modification carried out is mainly in the following three aspects: (1) Eliminating the inside of the ring one by one, and studying the contribution of each 1⁄2^t to the catalytic efficiency; (2) Using the natural 1⁄2 for each ring, the research is The effect of the absence of functional groups such as and the catalytic reaction; (3) the use of non-natural bases, such as 2-^ adenine and guanidine, etc. to increase or decrease the functional group, but most of its research scope is still limited to participate in Watson-Crick pairing. Functional basis.

In the screening of novel ribozymes, amino- and mito-modified nucleoside analogs have also been used, which are selected based on the cleavage mechanism of the nuclease. The use of these functional groups for acid-basic and hydrogen bonding, enhanced nucleophilicity of 2,-0, and promotion of protonation of 5,-oxyanions are the main strategies for accelerating this reaction. Nucleic acid 1⁄2 may play such a role, but under physiological pH conditions, its acidity and alkalinity are not optimal. Related prior art can be referred to the references attached hereinafter.

Therefore, the search for deoxyribozymes with higher catalytic performance is still expected in the art. Summary of the invention

The technical problem to be solved by the present invention is to find a 10-23 deoxyribozyme analog having higher catalytic efficiency. The invention mainly selects functional groups mainly composed of amino group, imidazolyl group and hydroxyl group, and introduces them into nucleoside monomers by various linkages, under a certain concentration of divalent metal ions and pH, 37 ° C By evaluating their contribution to catalytic activity at various sites in the catalytic domain of 10-23 deoxyribozyme, screening for more efficient deoxyribozymes, and studying the effects of these functional groups and spatial orientation on catalytic efficiency and exploring Possible catalytic mechanism. The invention utilizes a chemical modification method to modify 10-23 deoxyribozyme, and obtains a 10-23 deoxyribozyme analog having high catalytic efficiency higher than the prototype 10-23 deoxyribozyme under the approximate physiological Mg ²⁺ concentration. . The present invention has been completed based on the above findings.

Summary of invention

A first aspect of the invention provides a 10-23 deoxyribozyme analog partially modified by a 10-23 deoxyribozyme catalytic domain, which is represented by the formula:

3'- N! N ₂ N _{3 4} N ₅ N ₆ ······ Ν Χ Χ ₁₅ Χ ₁₄ <: ₁₃ X ₁₂ X _n C ₁₀ X ₉ X ₈ C ₇ XAX4

C3X2X1 j+17 j+18 i+i9 j+20 N _n -5' ,

Or as shown below:

N ₆ ...... Ni RN _i+17

i

"XX ₂

13C c ₃

₁₂ X x ₄

11X x ₅

10C x ₆

9X x ₈ c ₇

among them,

Representing the recognition moiety at both ends of the 10-23 deoxyribozyme analog, and the number of bases at both ends is the same or different, each independently from 4 to 25;

3'- X ₁₅ X ₁₄ C ₁₃ X ₁₂ XnC ₁₀ X9X8C7 X6X5X4 C3X2X1 -5, is the catalytic domain; n is an integer from 4 to 50;

i is an integer of 4-33;

15

14X x ₂

13C c ₃

₁₂ X x ₄

11X Xs

10C x ₆

The catalytic domain portion ₉ xx ₈ c ₇ is a _{1 (} )-23 deoxyribozyme catalytic domain portion

isA G,

14G G ₂

13C c ₃

12A τ ₄

11A A ₅

10C G ₆

Any one or more of the residues i, 2, 4, 5, 6, 8, 9, 11, 12, 14, 15 in ₉ A τ ₈ c ₇ are each independently selected from the following formula J , formula B,

J B D In the nucleoside analogs of the above formula J, formula 8, formula 0, formula E and formula F, each substituent is independently defined as follows:

(1) 嘌呤 nucleoside analogs J and D, each independently selected from carbon and nitrogen atoms, wherein

When ∑ is ^, R ^{1 in} position 7 is independently selected from: hydrogen, halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg cyano, thiophene), carboxyl, amide group

(eg CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ ), C _{6 2} . Aromatic group, C _{3 1} . a heteroaromatic and heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base, QM. Heteroaromatic and heterocyclic knots Structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )= NR ⁷ , halogen (eg fluorine, chlorine, bromine, nitrate), pseudohalogen (eg, sulfur, 1⁄2,

L is a Cwo straight or branched alkyl arm selected from the group consisting of

Linear or branched guanidine arms, such as methylene, 1,2-ethylene, trimethylene, tetramethylene), C ₂ _ _{1 ()} unsaturated alkyl arms (eg, olefinic, acetylenic) ), C ₃ .io arm cycloalkyl (e.g. C _3. ₆ cycloalkyl), it may be even further linear and branched structures containing an amide bond, an ester bond, an ether bond, a thioether bond,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a d- ₆ straight or branched alkyl group such as an anthracenyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group). , . Unsaturated alkyl (e.g., c _2. ₄ unsaturated alkyl, such as vinyl, prop cockroach group, ethynyl group, propynyl group),

. Cycloalkyl group (e.g., c _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, the aromatic group and the heteroaromatic optionally substituted with one or more substituent groups R ^9, R ⁹ the same definition as R ^7;

(2) The 2-position substituents R ^{2 of the} purine nucleoside analogs J, B, D, E are each independently selected from the group consisting of: hydrogen, hydroxy, cyclin (e.g., fluorine, chlorine, bromine, reef), sulfhydryl, OR ⁷ ^{^{.., NHR 7, NR 7}} 2, NHCOR 7 ( amine group), a mercapto group, SR ^7, C ₆ ₂ o aryl, C ₃ ₈ heteroaromatic and heterocyclic structure, R ^7, or LR ^8, wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ _ ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ > NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), St&, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ )=NR ⁷ halogen (eg fluorine, chlorine, bromine, ), pseudohalogen

(eg, sulfur n^), 1⁄2,

L is a connecting arm selected from the following: Linear or branched alkyl arm (eg d.6) A linear or branched alkyl arm such as anthracenylene, 1,2-ethylene, trimethylene, tetramethylene). An unsaturated alkyl arm (eg, an olefinic bond, an acetylenic bond), a C ₃ .io cycloalkyl arm (eg, a C ₃ _-6 cycloalkyl group), which may also be an amide-containing bond, an ester bond, an ether bond, or a sulfur a linear and branched structure of an ether bond,

R ^{7 is} each independently selected from: C" ₀ straight or branched alkyl (for example, d.6 straight or branched alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, iso Butyl, tert-butyl, pentyl, hexyl), C ₂ _.10 unsaturated fluorenyl (eg C ₂ _ ₄ unsaturated alkyl, eg vinyl, propenyl, ethynyl, propynyl), C ₃ . Io cycloalkyl (for example, C ₃ _-6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, said aromatic and heteroaromatic optionally substituted with one or more substituents substituted with R ^9, R ⁹ the same definition as R ^7;

(3) The substituents R ^{3 of the} purine nucleoside analogs J and B are each independently selected from the group consisting of: hydrogen, amino, hydroxyl, halogen (e.g., fluorine, chlorine, bromine, iodine), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, St &, SR ⁷ , C ₆ _ _{2 ()} aryl, C ₃ . ₂₀ heteroaryl and heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), 53⁄4J^, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH- C(NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg, sulfur, ,

L is a link selected from the following. Linear or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms such as fluorenylene, 1,2-ethylene, trimethylene, tetramethylene), C ₂ _ ₁₍₎ An unsaturated alkyl arm (such as an olefinic bond, an acetylenic bond), a C ₃ -io cycloalkyl arm (for example, a C ₃ _-6 cyclodecyl group), and the linking arm may also be an amide-containing bond, an ester bond, an ether bond, or a thioether. The linear and branched structure of the bond,

R ^{7 is} each independently selected from. Linear or branched alkyl (eg C straight or branched) Chain pit bases, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl). Unsaturated alkyl (e.g., C _2. ₄ unsaturated alkyl group, e.g. cockroach ethyl group, propyl group cockroach, acetylene, propynyl group), c _w. Cycloalkyl group (e.g., c _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, purine nucleoside analogs J and D The 8 bits of W may each independently be a carbon atom or a nitrogen atom, wherein:

When w is, neutrons, which is optionally substituted with R ^{1 ()} substituent, the R ¹⁰ taken independently selected from: hydrogen, halo (e.g. fluoro, chloro, bromo, iodo), pseudohalogen (e.g., sulfur ^ J^), COOR ⁷ (ester group), CONH ₂ , CONHR ⁷ , CO R ⁷ ₂ (amide group), J^, fluorenyl group, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl group), 5t&, SR ⁷ , C ₆ _ ₂ . Aromatic base. a heteroaromatic and heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), 3⁄43⁄4JL, SR ⁷ > CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (R ⁷ ₂ )= R ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg thiocyano), hydrazine

L is a connection selected from the following _: a linear or branched arm (for example, a d- ₆ straight or branched alkyl arm such as an anthranylene group, a 1,2-ethylene group, a triadenylene group, a tetradecyl group). Arm unsaturated alkyl (e.g., ethylenic, acetylenic _bond), C _3. _{1 ()} arm cycloalkyl (e.g. C _3. ₆ cycloalkyl arm), the connecting arm may also contain an amide bond, an ester bond, an ether bond , a linear and branched structure of a thioether bond,

R ^{7 is} each independently selected from a linear or branched alkyl group (eg,

Linear or branched alkyl group, such as methyl, ethyl, propyl, iso Propyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl), C ₂ _.10 unsaturated alkyl (eg C ₂ _ ₄ unsaturated alkyl, such as vinyl, propenyl, ethyl, propyl Alkynyl), c ₃ . ₁₍₎ cycloalkyl

(for example, C ₃ -6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring,

The aryl and heteroaryl are optionally substituted by one or more substituents

R ^{9 is} substituted, and R ^{9 is} the same as R ⁷ ;

When W is a nitrogen atom, it is not substituted;

The five-membered ring of adenosine analogues B and E, Z and V is a saturated ring structure, and Z and V are each independently an atom of carbon, nitrogen, oxygen, sulfur, etc.

When ∑ is ^, R ^{1 is} independently selected from the group consisting of: hydrogen, halogen (eg, fluorine, chlorine, bromine, iodine), pseudohalogen (eg, sulfur · 1⁄2), silk, amide (eg, CONH) ₂ , CONHR ⁷ , CONR ⁷ ₂ ), C ₆ _ ₂ . Aromatic group, C ₃ . ₂ . a heteroaromatic or heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of hydroxyl, C ₆ _ ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , R ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , CONH ₂ , CONH ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg cyano, thiocyano), carboxyl group,

L is a link selected from the following. a linear or branched alkyl arm (for example, a d- ₆ straight or branched alkyl arm such as methylene, 1,2-ethylene, trimethylene, tetramethylene), C ₂ _.1() An unsaturated alkyl arm (such as an olefinic bond, an acetylenic bond), a C ₃ _ _{1 ()} cycloalkyl arm (for example, a C ₃ -6 cycloalkyl arm), and the linking arm may also be an amide-containing bond, an ester bond, or an ether bond. , a linear and branched structure of a thioether bond,

R ^{7 is} each independently selected from. Linear or branched alkyl (eg CM Linear or branched pit bases such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl). Unsaturated alkyl (e.g., C ₂ ₄ unsaturated alkyl groups, such as vinyl, propenyl, B;. ^, _Propynyl), c ₃ _ _{1 ()} cycloalkyl (e.g., c ₃ _ ₆ cycloalkyl, For example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), as well as linear and branched structures containing aromatic rings,

The aryl group and heteroaryl group are optionally substituted by one or more substituents R ⁹ , and R ^{9 has} the same meaning as R ⁷ ;

When Z is a nitrogen atom, it is taken as R ¹ , but does not include halogen and pseudohalogen;

When ¥ is a subunit, each of them is optionally independently substituted with J J R ¹¹ , and the definition of the substituent R ^{11 is} the same as R ¹ ;

When V is a nitrogen atom, it has the same definition as R ¹ but is not substituted by halogen and pseudohalogen.

When Z and V are oxygen or sulfur atoms, there is no substituent;

The pyrimidine nucleoside analog F, wherein the substituents R ⁴ and R ^{5 at the 5-} and 6-positions are each independently selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, iodine), J^, fluorenyl, pseudohalogen (H^ , sulfur HS^), ^^, COOR ⁷ (ester group), CO H ₂ , CONHR ⁷ , CONR ⁷ ₂ (amide group), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCO ⁷ (aminoacyl), 51⁄2 SR ⁷ , C ₆ _ ₂ . Aromatic and. a heteroaromatic and heterocyclic structure, R ⁷ , or LR ⁸ , wherein: R ^{8 is} selected from the group consisting of hydroxyl, amino, C ₆ _ ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH- C(NR ⁷ ₂ )=NR halogen (eg fluorine, chlorine, bromine, ), pseudohalogen (eg sulfur,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a d. ₆ linear or branched pit group such as an anthracenyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, or a different group) Butyl, tert-butyl, pentyl, hexyl), . An unsaturated alkyl group (eg, a C ₂ _4 unsaturated alkyl group such as a vinyl group, a propenyl group, an ethyl group, a propyl group),

. Cycloalkyl (e.g. C _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring,

L is selected even dH) a straight-chain or branched-chain alkyl group arms (e.g. d_ ₆ linear or branched alkyl arm, such as an alkylene group Yue, 1, 2-ethylidene, trimethylene Yue group, tetramethylene Base), . An unsaturated alkyl arm (such as an olefinic bond, a block bond), a C ₃ .io cycloalkyl arm (for example, a C ₃ _-6 cyclopentyl arm), and the linking arm may also be an amide bond, an ester bond, an ether bond, or a sulfur linear and branched structures ether bond, an aromatic group and the heteroaromatic group optionally substituted with one or more substituents substituted with R ^9, R ⁹ the same definition as R ^7;

(7) nucleoside analogs J, B, D, E, F, the sugar ring portions of which are each independently selected from the group consisting of ribosyl, deoxyribose, other five-membered sugar ring, six-membered sugar ring, LNA type, or Other modified sugar ring structures, each of which is independently of a D- or L-form, wherein

When the sugar ring moiety is a five member sugar ring, the 2,-position substituents R ⁶ are each independently selected from hydrogen,

,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Ethylamino, propylamino, dipropylamino, cyclopropylamino. According to a first aspect of the present invention, in particular, a 10-23 deoxyribozyme analog having a 10-23 deoxyribozyme catalytic domain partially modified is provided,

It is as follows:

3'- Ni N ₂ N ₃ N ₄ N ₅ N ₆ ······ Ν 〖 X ₁₅ X ₁₄ C ₁₃ X, ₂ XiiC ₁₀ X ₉ X ₈ C ₇

C3X2X1 RN _i+17 N _i+18 N _i+19 N _i+20 ······ N _n -5,,

Or the following two-dimensional structure when combined with a substrate: 5'- ΝΊ Ν'2 Ν'3 Ν' ₄ Ν' ₅ Ν, ₆ Υ Ν' _ί+3 N' _i+ 4 N'i ₊₅ N' _i+6 Ν' m _3,

among them,

From ,-? ^ to , -! ^ is the nucleic acid substrate sequence, m is the number of substrate monomers; from 3,-]^ to 5,-]\„ is the recognition domain of the deoxyribozyme, n is its number; X is the introduced ^ "decorated nucleotide monomer; R is a fluorene monomer, and Y is a pyrimidine monomer;

N, which is the target sequence composition of deoxyribozyme, 5,- Ν N ₂ , N ₃ , N ₄ , N, ₅ N, ₆ ······· Ni, RY

N, _i+3 N, _i+4 N, _i+5 N, _i+6 ······ N, _m -3, is the nucleic acid substrate sequence of the 10-23 deoxyribozyme analog, wherein R A purine nucleotide monomer, Y is a pyrimidine nucleotide monomer, and a plurality of N, the sequence consisting of a fragment selected from any gene or a full-length gene, m≥4. The arrow points to the cleavage site;

N represents the recognition moiety at both ends of the 10-23 deoxyribozyme analog, the number of bases at both ends being the same or different, each independently from 4 to 25; and the partial sequence at both ends of the cleavage site in the sequence of the nuclear sequence Paired with Watson-Crick.

3'- Xi ₅ X ₁₄ C ₁₃ Xi ₂ XiiC ₁₀ X ₉ X ₈ C ₇ X6X ₅ X4 C ₃ X ₂ Xi -5' is a catalytic domain; wherein X is a modified nucleomonomer and C is a base a nucleomonomer nucleomonomer, R is a purine nucleotide monomer;

n is an integer from 4 to 50;

i is an integer from 4 to 33; 1ϋΧ Xi

x ₂

13C c ₃

₁₂ x

11X x ₅

10C x ₆

The catalytic domain portion ₉ xx ₈ c ₇ is a 10-23 deoxyribozyme catalytic domain

15A G1

14G G ₂

13C c ₃

12 ^A T ₄

1lA A ₅

10C G ₆

Any one or more of the residues 2, 4, 5, 6, 8, 9, 11, 12, 14, 15 of ₉ A τ ₈ c ₇ are each independently selected from the following formula J, Formula B,

(1) The quinone nucleoside analogs J and D, the atom Z at the 7 position may be independently selected from carbon and nitrogen atoms. among them,

When ∑ is ^, the 7 positions on R ¹ are each independently selected from: hydrogen, halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen (eg, sulfur, silk, amide group (eg CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ ), C ₆ . ₂₀ aromatic, C ₃ _.10 heterocyclic or heteroaryl, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ _ ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ > NHCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )= R ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg bismuth sulphur, antimony)

L is a link selected from the following. A linear or branched alkyl arm (for example, a d- ₆ straight or branched arm such as methylene, 1,2-ethylene, trimethylene, tetradecyl). Arm unsaturated alkyl (e.g., ethylenic, acetylenic bond), C ₃ .io arm cycloalkyl (e.g. C _3. ₆ cycloalkyl), containing the connecting arm may also be an amide bond, an ester bond, an ether bond, a thioether The linear and branched structure of the bond,

R ^{7 is} each independently selected from the group consisting of dm linear or branched saturated alkyl groups and unsaturated alkyl groups (for example, d.6 straight or branched alkyl groups such as propyl, isopropyl, n-butyl, isobutyl) , tert-butyl, pentyl, hexyl), . An unsaturated alkyl group (for example, a C ₂ _ ₄ unsaturated alkyl group such as an ethyl hydrazino group, a propyl fluorenyl group, an ethynyl group, a propyl group). Cycloalkyl group (e.g., c _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, the aromatic group and the heteroaromatic optionally substituted with one or more substituent groups R ^9, R ⁹ the same definition as R ^7;

When Z is a nitrogen atom, there is no substitution;

The 2-position substituents R ^{2 of the} purine nucleoside analogs J, B, D, E are each independently selected from the group consisting of: hydrogen, hydroxy, halogen (eg, fluorine, chlorine, bromine, iodine), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, silk, SR ⁷ , C ₆ - ₂₀ aryl, C ₃ _ ₁₀ heterocyclic or heteroaryl, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl HC (NR ⁷ ₂ ) = NR halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen

(eg L^, sulfur ^ J, 纮

L is a tether selected from the group consisting of: dm linear or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms such as anthracenylene, 1,2-ethylene, trimethylene, _{Tetramethylene),} C ₂ _ _{1 ()} arm unsaturated alkyl (e.g., ethylenic, acetylenic bond), C _3., ₀ cycloalkyl arm (e.g. C _3. ₆ cycloalkyl), can also be connected to the arm Is a linear and branched structure containing an amide bond, an ester bond, an ether bond, or a thioether bond.

R ^{7 is} each independently selected from: a linear or branched saturated alkyl group (for example, a C _M straight or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl,己基), . An unsaturated alkyl group (for example, an unsaturated alkyl group such as a vinyl group, a propenyl group, an ethynyl group, a propyl group), a c ₃ _.1() cycloalkyl group (for example, a c ₃ _-6 cycloalkyl group such as a cyclopropyl group, Cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing aromatic rings,

The substituents R ^{3 of the} purine nucleoside analogs J and B are each independently selected from the group consisting of: hydrogen, amino, hydroxy, halogen, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , HCOR ⁷ (aminoacyl), sulfhydryl, sparingly Base, SR ⁷ , C ₆ _ ₂ . Aromatic base. a heterocyclic or heteroaromatic group, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and a C ₆ — _2e aromatic group. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C ( NR ⁷ ₂ )=NR\ Halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen

(eg 纮 sulfur, ,

L is a connecting arm selected from the following: Linear or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms such as fluorenylene, 1,2-ethylene, trimethylene, tetramethylene), C ₂ _ ₁₍₎ An unsaturated alkyl arm (e.g., an olefinic bond, an acetylenic bond), a C ₃ .iocyclononyl arm (e.g., a C ₃ _-6 cycloalkyl group), which may also be an amide bond, an ester bond, an ether bond, or a thioether bond. Straight and branched structure,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, d. ₆ straight chain or Branched alkyl, for example, decyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl), C ₂ .io unsaturated alkyl (eg C ₂ . ₄ unsaturated alkyl groups, such as vinyl, propenyl, ethynyl, propynyl). Cycloalkyl (e.g. C _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, purine nucleoside analogs J and D The 8 bits of W may each independently be a carbon atom or a nitrogen atom, wherein:

When W is a carbon atom, it is optionally substituted by a substituent R ¹ ", each independently selected from: hydrogen, halogen (eg, fluorine, chlorine, bromine, ), pseudohalogen (eg, cyano, thiocyano), carboxy , COOR ⁷ (ester group), CO H ₂ , CONHR ⁷ , CONR3⁄4 amide group), amino group, sulfhydryl group, OR ⁷ , HR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl group), fluorenyl group, SR ⁷ , C ₆ . _{2. An} aromatic group, a C ₃ _.1() heterocyclic or heteroaryl group, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), H SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ )= R ⁷ , halogen (eg fluorine, chlorine, bromine, angstrom), pseudohalogen (eg ^, sulphur, silk,

L is a tether selected from the group consisting of: C _M o linear or branched alkyl arms (eg, d.6 linear or branched alkyl arms such as anthracenylene, 1,2-ethylene, trimethylene) , tetramethylene), C ₂ _{1 ()} unsaturated alkyl arms (eg, olefinic bonds, acetylenic bonds), C ₃ .io cycloalkyl arms (eg, C ₃ ₆ cycloalkyl), even ^^ It may be a linear or branched structure containing an amide bond, an ester bond, an ether bond, or a thioether bond.

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a linear or branched alkyl group of d. ₆ such as an anthracenyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group; ), . An unsaturated alkyl group (for example, a c ₂ _ ₄ unsaturated alkyl group such as an ethyl group, a propyl group, an ethynyl group, a propynyl group), . Cycloalkyl (e.g. C _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, the aromatic group and the heteroaromatic optionally substituted with one or more substituent groups R ^9, R ⁹ the same definition as R ^7;

When W is a nitrogen atom, it is not substituted;

In the purine nucleoside analogs B and E, the five-membered ring in which Z and V are located is a saturated ring structure, and Z and V are each independently an atom such as carbon, nitrogen, oxygen, sulfur, etc.

When it is a substrate, the substituents R ¹ thereon are each independently selected from the group consisting of: hydrogen, halogen (e.g., fluorine, chlorine, bromine, argon), pseudohalogen (e.g., *J^, sulfur^J^), amide Base (eg, CONH ₂ , CONHR ⁷ > CONR ⁷ ₂ ), C _6. ₂₀ aryl, . a heterocyclic or heteroaryl group, R ⁷ , or R ⁸ wherein: R ^{8 is} selected from the group consisting of hydroxyl, amino, C ₆ _ ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , CO H ₂ , CONHR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl HC (NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg H group, thiocyano), carboxyl group,

L is a tether selected from the group consisting of: C _M0 linear or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms, such as anthracenylene, 1,2-ethylene, trimethylene, tetra Amidinoyl), C ₂ _ _1G unsaturated alkyl arms (eg, olefinic bonds, acetylenic bonds). a cycloalkyl arm (for example, a C ₃ -6 cycloalkyl group), the linking arm may also be a linear or branched structure containing an amide bond, an ester bond, an ether bond, or a thioether bond.

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a linear or branched alkyl group of d. ₆ such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl ), . Unsaturated alkyl (e.g., C _2. ₄ unsaturated alkyl, such as vinyl, propenyl, ethynyl, _propynyl), c ₃ _ _{1 ()} alkyl with cyclic (e.g. c ₃ _ ₆ cycloalkyl, For example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), as well as linear and branched structures containing aromatic rings,

When V is ^^, it takes R ¹¹ and its definition is the same as R ¹ .

When V is a nitrogen atom, each of them is optionally independently substituted with R ¹¹ ,

R ^{11 has} the same definition as R ¹ but is not substituted by halogen and pseudohalogen; when z and V are oxygen or sulfur atoms, there is no radical;

The pyrimidine nucleoside analog F, wherein the substituents R ⁴ and R ^{5 at} positions 5 and 6 are each independently selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, reef), pseudohalogen (, sulfur), COOR ⁷ ( Ester group), CONH ₂ , CONHR ⁷ , CO R ⁷ ₂ (amido group), J^, fluorenyl group, NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), SR ⁷ , C ₆ _-20 aromatic group and . Heterocyclic or heteroaryl, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base, Q. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), SR ⁷ , CONH ₂ , CONHR ⁷ , CO R ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C ( NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg ^, sulfur, ,

R ^{7 is} each independently selected from a linear or branched alkyl group (for example, a C straight or branched chain group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl) , amyl, hexyl), . An unsaturated alkyl group (for example, a C ₂ _ ₄ unsaturated alkyl group such as an ethyl group, a propyl group, an ethyl group, a propynyl group),

C ₃ _ _{1 ()} cycloalkyl (e.g. C _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched aromatic ring-containing citrate link,

L is a linear or branched alkyl arm selected from the group consisting of: C _M0 (for example, d. ₆₎ A linear or branched alkyl arm such as an anthracenylene group, a 1,2-ethylene group, a triadenylene group, a tetramethylene group, or the like. An unsaturated alkyl arm (e.g., an olefinic bond, an acetylenic bond), a C ₃ -io cycloalkyl arm (e.g., a C ₃ _-6 cycloalkyl group), which may also be an amide-containing bond, an ester bond, an ether bond, or a a linear and branched structure of an ether bond,

(7) nucleoside analogs J, B, D, E, F, the sugar ring portions of which are each independently selected from the group consisting of ribosyl, deoxyribose, other five-membered sugar ring, six-membered sugar ring, LNA type, or Other modified sugar ring structures [preferably, the sugar ring portions are each independently selected from the group consisting of deoxyribose groups, LNA types], and the configuration of the sugar ring portions are each independently D- or L-type, wherein

When the sugar ring moiety is a five member sugar ring, the 2,-position substituents R ⁶ are each independently selected from the group consisting of hydrogen, ^, a, ethoxy, and propyl! ^, methoxy ethoxylate | L &, b ft-vinyl oxime, propyl (glycol fluorenyl, decylamino, dimethylamino, ethylamino, diethylamino, propylamine, dipropylamine A cycloalkanoamine analog according to any one of the first aspects of the present invention, wherein Ν represents a recognition moiety at both ends of the 10-23 deoxyribozyme analog, and the number of bases at both ends may be the same, The 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein Ν represents an identification moiety at both ends of the 10-23 deoxyribozyme analog, and the number of 51⁄2 at both ends is different. According to the present invention A 10-23 deoxyribozyme analog according to any one of the preceding claims, wherein Ν represents the recognition moiety at both ends of the 10-23 deoxyribozyme analog, and the number of the two ends is the same or different, each independently from 4 to 25 .

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein n is an integer from 4 to 50. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein n is an integer from 10 to 40. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein n is an integer from 15 to 40. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein n is an integer from 20 to 40. First party according to the invention Any of the 10-23 deoxyribozyme analogs, wherein n is an integer from 4 to 40.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein i is an integer from 4 to 33. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein i is an integer from 4 to 25. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein i is an integer from 4 to 12. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein i is an integer from 6 to 12.

A 10-23 deoxyribozyme analog according to any one of the preceding aspects, wherein said catalytic domain portion is the first, second, fourth, fifth, and sixth portions of the 10-23 deoxyribozyme catalytic domain portion. Any of residues 8, 9, 11, 12, 14, or 15 is substituted with a nucleoside analog selected from Formula J, Formula B, Formula 0, Formula, and Formula F. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is the 1, 2, 4, 5, 6 of the 10-23 deoxyribonucleotide domain portion. Any two or more of the residues 8, 8, 9, 12, 14, or 15 are substituted with a nucleoside analog selected from the group consisting of Formula J, Formula 8, Formula 0, Formula E, and Formula F.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is 5, 9, 11, 12, or in the 10-23 deoxyribozyme catalytic domain portion Any of the residues No. 15 is substituted with a nucleoside analog selected from the group consisting of Formula J, Formula 8, Formula 0, Formula E, and Formula F. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is the 1, 2, 6, or 14 of the 10-23 deoxyribozyme catalytic domain portion Any of the residues is substituted with a nucleoside analog selected from the group consisting of Formula J, Formula 0, Formula E, and Formula F. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is any of residues 4 or 8 in the 10-23 deoxyribozyme catalytic domain portion One is substituted with a nucleoside analog selected from Formula J, Formula B, Formula D, Formula E, and Formula F.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is 5, 9, 11, 12, or in the 10-23 deoxyribozyme catalytic domain portion Any of the residues No. 15 is substituted with a nucleoside analog selected from the group consisting of Formula J and Formula B. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is the 1, 2, 6, or 14 of the 10-23 deoxyribozyme catalytic domain portion Any of the residues is substituted with a nucleoside analog selected from Formula D and Formula E. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is any of residues 4 or 8 in the 10-23 deoxyribozyme catalytic domain portion One is substituted with a nucleoside analog selected from Formula F.

(1) A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein said catalytic domain portion is 5, 9, 11 in the 10-23 deoxyribozyme catalytic domain portion. Any of residues 12, or 15 is substituted with a nucleoside analog selected from the group consisting of:

.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is the 1, 2, 6, or 14 of the 10-23 deoxyribozyme catalytic domain portion Any of the residues are substituted with a nucleoside analog selected from the group consisting of: .

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic domain portion is a 10-23 deoxyribozyme catalytic domain 4 or 8

Any of the residues are substituted with a nucleoside analog selected from the group consisting of:

R ⁴ selection

R ⁴ = CH ₂ OH

R ⁴ = CH ₂ CH ₂ OH

R ⁴ = CH ₂ CH ₂ CH ₂ OH

From: R ⁴ = CH ₂ CH ₂ (4-) lm.

(2) A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein in the nucleoside analog of the formula J and the formula D, the atom Z at the 7 position may be independently selected From carbon and nitrogen atoms. among them,

When z is ^^ midnight, 7 on which either R ¹ is independently selected from: hydrogen, halogen (e.g. fluorine, chlorine, bromine, iodine), a pseudohalogen (e.g. cyano, thiocyano), Q. 20 an aromatic group (for example, a C ₆ aromatic group such as a phenyl group). Heterocyclic or heteroaryl group (e.g. imidazolyl, pyrazolyl ^),, R ^7, or LR ^8, wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ _ ₂ . An aromatic group (for example, a C ₆ aromatic group such as a phenyl group). Heterocyclic or heteroaryl (eg imidazolyl, pyridyl), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )= R ⁷ , fluorenyl, SR ⁷ , CO H ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen (eg cyano, sulphur ^ J , ,

L is a combination of the following ^f: . Straight or branched; ^ arm (for example

Linear or branched alkyl arms, such as methylene, 1,2-ethylene, trimethylene, tetradecyl). An unsaturated alkyl arm (eg, a C ₂ _ ₄ unsaturated alkyl arm, such as an olefinic bond, a block bond), . Arm cycloalkyl (e.g. C _3. ₆ cycloalkyl arm),

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a C straight or branched alkyl group such as an anthracenyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group), c ₂ _ _{1 ()} unsaturated alkyl (e.g., c _2. ₄ unsaturated alkyl, such as vinyl, propenyl, ethynyl, propynyl),

. Ring-entangled groups (e.g., c ₃ _-6 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl). And linear and branched structures containing an aromatic ring, such as benzyl, phenethyl, tolylethyl, tert-butylphenethyl, phenylpropyl and the like. When Z is a nitrogen atom, there is no substituent.

10-23 DNAzyme analogue according to any of the first aspect of the present invention, wherein the Formula J, Formula Formula 0, nucleoside analogues of formula E, the 2 substituents R ² are each independently selected from : hydrogen, hydroxy, halogen (eg fluorine, chlorine, bromine, reef), C ₆ _-20 aromatic, C ₃ _ _{1 ()} heterocyclic or heteroaromatic, fluorenyl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , HCOR ⁷ (aminoacyl), thiol, SR ⁷ , R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , HR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ ,

CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen (eg cyano, sulphur, ,

L is a tether selected from the group consisting of Cwn straight or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms such as anthracenylene, 1,2-ethylene, trimethylene, tetra Methyl), . An unsaturated alkyl arm (eg, an ethylenic bond, a block bond), a C ₃ .io cycloalkyl arm (eg, a C ₃ _-6 cycloalkyl group),

R ^{7 is} each independently selected from: Cwo straight or branched alkyl (for example, d. ₆ straight or branched alkyl such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl , tert-butyl, pentyl, hexyl), C ₂ _ _{1 ()} unsaturated alkyl (eg C ₂ 4 unsaturated alkyl such as vinyl, propenyl, ethynyl, propyl), C ₃ .io A cycloalkyl group (e.g., a C ₃ _-6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group). And linear and branched structures containing an aromatic ring, such as benzyl, phenethyl, decyloxyphenethyl, tert-butylphenethyl, phenylpropyl and the like. When Z is a nitrogen atom, then no l group is taken. 10-23 DNAzyme according to any one of the analogs of the first aspect of the present invention, wherein said compound of formula J, nucleoside analogues of formula B, the 6-substituted group R ³ is independently selected from: hydrogen, J ^, hydroxy, ^{^{halogen, OR 7, NHR 7, NR}} 7 2, NHCOR 7 ( amine group), guanidino, ^{_{_{thiol, SR 7, C 6. 2}}} . Aromatic base. Heterocyclic or heteroaromatic, R ⁷ , LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted ^ I fluorenyl NH-C(R ⁷ ₂ )=NR ⁷ , SftJ^ SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ halogen (such as fluorine, chlorine, bromine, hydrazine), pseudo-halogen (for example, Sulfur ^), silk,

L is a connecting arm selected from the following: A linear or branched alkyl arm (for example, a d- ₆ straight or branched alkyl arm such as methylene, 1,2-ethylene, trisino, tetramethylene). Arm unsaturated alkyl (e.g., ethylenic, acetylenic bond), C ₃ .io arm cycloalkyl (e.g. C _3. ₆ cycloalkyl), containing the connecting arm may also be an amide bond, an ester bond, an ether bond, a thioether The linear and branched structure of the bond,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a d- ₆ straight or branched alkyl group such as decyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl) , C ₂ .io unsaturated alkyl (for example

C ₂ _ ₄ unsaturated alkyl group, such as vinyl, propenyl, ethynyl, propynyl). a cycloalkyl group (for example, a C ₃ _-6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group), and a linear and branched structure containing an aromatic ring, such as a benzyl group, a phenethyl group, Toluene ethyl, tert-butylphenethyl, phenylpropyl and the like. A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein in the nucleoside analog of the formula J, formula D, the 8 positions W may each independently be a carbon atom or a nitrogen atom, wherein :

When w is midnight ^^, optionally substituted with a substituent R ^IE, taking the R ¹⁰ is independently selected from: hydrogen, halogen (e.g. fluorine, chlorine, bromine, change), C ₆ ₂₀ aryl. Heterocyclic or heteroaromatic, silk, COOR ⁷ (ester), CONH ₂ , CONHR ⁷ , CO R ⁷ ₂ (amido), amino, sulfhydryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (amine Acyl), R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , Sulfhydryl, substituted fluorenyl NH-C(R ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg ^, thiocyano), hydrazine

L is a link selected from the following _: C ₁₄ . a linear or branched alkyl arm (eg, a straight or branched alkyl arm such as methylene, 1,2-ethylene, trimethylene, tetramethylene), C ₂ _ ₁₍₎ arm saturated alkyl (e.g. cockroach bond, acetylene bond), C ₃ _ _ie arm cycloalkyl (e.g. C _3. ₆ cycloalkyl arm),

R ^{7 is} each independently selected from. Linear or branched alkyl (eg

a linear or branched alkyl group, for example, anthracenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl),

. Unsaturated alkyl (e.g., c ₂ _ ₄ unsaturated alkyl groups, such as vinyl, propenyl, B;. ^, Propoxy ^ ¾, c ₃ ₁₀ cycloalkyl group (e.g., c ₃ ₆ cycloalkyl, cyclopropylmethyl e.g. a base, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group, and a linear and branched structure containing an aromatic ring, such as a benzyl group, a phenethyl group, a methoxyphenethyl group, a t-butyl group ethyl group Base

When W is a nitrogen atom, there is no substituent.

A 10-23 deoxyribozyme analog according to any one of the preceding claims, wherein in the nucleoside analog of formula B, formula E, Z is each independently a carbon atom and the substituent is R.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein in the nucleoside analog of the formula B, formula E, each V is independently a carbon atom or a nitrogen atom, which is optionally Substituent R ¹¹ substituted;

When V is a moiety, R ^{11 is} each independently selected from the group consisting of: hydrogen, halogen (eg, fluorine, chlorine, bromine, ), pseudohalogen (eg, an aryl group, a thiol group), a carboxyl group, an amide group (eg, CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ ), . Aromatic base. Heterocyclic or heteroaromatic, R ⁷ , or LR ⁸ , where:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . Aromatic base. Heterocyclic or heteroaryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , CONH ₂ , CO HR ⁷ , CONR ⁷ ₂ , fluorenyl, substituted fluorenyl NH- C(NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg, sulfur ^ J , ,

L is a tether selected from the group consisting of: C _w . a linear or branched alkyl group (for example, a d- ₆ straight chain or a branched group; for example, a methylene group, a 1,2-ethylene group, a trimethylene group, a tetramethylene group). Unsaturated alkyl arms (e.g. arm unsaturated alkyl, e.g. ethylenic, acetylenic bond), C ^ cycloalkyl arm (e.g. C _3. ₆ cycloalkyl arm),

R ⁷ are each independently selected from Cwo straight or branched alkyl groups (eg straight or branched alkyl groups such as decyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl) , amyl, hexyl), . Unsaturated alkyl (e.g., c _2. ₄ unsaturated alkyl, such as vinyl, propenyl, ethynyl, _propynyl), c _3. _{1 ()} cycloalkyl (e.g., c _3. ₆ cycloalkyl, e.g. Cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl). And linear and branched structures containing an aromatic ring, such as benzyl, phenethyl, methoxyphenethyl, tert-butylphenethyl, phenylpropyl and the like.

When V is a nitrogen atom, they are each independently optionally substituted by R ¹¹ , which has the same definition as R ¹ but is not halogen and pseudohalogen; 10-23 deoxygenation according to any of the first aspects of the invention a ribozyme analog, wherein in the nucleoside analog of the formula F, the substituents R ⁴ and R ^{5 at the 5-} and 6-positions are each independently selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, iodine), Pseudohalogen (#J^, sulfur #J, ^&, COOR ⁷ (ester group), CONH ₂ , CO HR ⁷ , CONR ⁷ ₂ (amide group), J^, sulfhydryl, NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , C ₆ . ₂ . an aromatic group (for example, a C aryl group such as a phenyl group), a heterocyclic ring or a heteroaryl group (for example, a C ₃ _-8 heteroaromatic group such as an imidazolyl group, Pyridyl), R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ . ₂ . An aromatic group (for example, a C ₆ aromatic group such as a phenyl group). Heterocyclic or heteroaryl (e.g., C ₃ _ ₈ heteroaryl, such as imidazolyl, pyr OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ )= R ⁷ , silk, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, ), pseudohalogen (eg, sulfur HJ, ,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a C straight or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl), C ₂ _.1() unsaturated thiol (eg, C ₂ _ ₄ unsaturated alkyl group, such as ethenyl, propenyl, ethynyl, propynyl),

C _M . Cycloalkyl group (e.g., c _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl). And linear and branched structures containing an aromatic ring, such as benzyl, phenethyl, methoxyphenylethyl, tert-butylphenethyl, phenylpropyl and the like.

L is a CMO linear or branched alkyl arm selected from the group consisting of: a d- ₆ straight or branched alkyl arm such as an anthranylene group, a 1,2-ethylene group, a triadenylene group, or a tetra Methylene), . Unsaturated alkyl arms (eg, olefinic bonds, acetylenic bonds), . Arm cycloalkyl (e.g. C _3. ₆ cycloalkyl arm), 10-23 deoxyribozyme like according to a first aspect of the present invention, any one, of formula wherein J, type 8, type 0, type Σ And a nucleoside analog of formula F, wherein the sugar ring moieties are each independently selected from the group consisting of deoxyribose, other five member sugar ring groups, other six member sugar ring groups, LNA type [preferably, the sugar ring portions are each independently It is selected from the group consisting of deoxyribose and LNA.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein the nucleoside analogs of the formula J, formula B, formula D, formula E and formula F have their sugar ring moieties independently Desirably selected from the group consisting of deoxyribosyl groups, LNA type, wherein the 2,-position substituents R ⁶ are each independently selected from the group consisting of hydrogen, a gas group, a fluorine atom, a decyloxy group, an ethoxy group, a propyl group, a propyl group, a decyloxy group. Base, B Ethylene L^, vinylidene HJ^.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, which is based on chemical modification of the catalytic domain of 10-23 deoxyribozyme to obtain a more efficient deoxyribozyme having a structure of 3,- N! N ₂ N ₃ N ₄ N ₅ N ₆ ·"··· Ni X ₁₅ X ₁₄ C ₁₃ X ₁₂ X„C ₁₀ X ₉ X ₈ C ₇

G ₃ X ₂ Xi RN _i+17 N _i+18 N _i+19 N _i+20 ······ N _n -5' „ R is a purine base, which is complementary to Y of the target sequence cleavage site; Ν represents the identification part at both ends, and the number of 1⁄2 at both ends is the same or different, ranging from 4 to 25. The structure of the target sequence recognized by it is 5, - ΝΊ Ν ' ₂ Ν, ₃ Ν, ₄ ··" _{·· N, i RY N 'i} + 3 N, i + 4 N' i + 5 N 'i + 6 ······ N, m -3 ,, R is a purine ½,

Y is pyrimidine-like, 5,-RY-3, which is the cleavage site of deoxyribozyme, and the two ends are sequences recognized by oxyribozymes. The length of the target sequence can range from 4 to the total number of gene sequences. Etc., they can be fragments of genetic manipulation or disease-causing genes. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, intermediate 3,-X ₁₅ X ₁₄ C ₁₃ XuXudo X ₉ X8C ₇ X6X ₅ X4 C3X2X1 -5' is a catalytic domain, X represents repair饰 nucleoside analogues, its design: ^ 下:

5'- ΝΊ N'2 N'3 N' ₄ N' ₅ N' ₆ ...... ΝΊ R Ύ N' _i+3 N' _i+4 N' _i+5 N' _i+6 .. .... N' _m -3'

3'- ! N ₂ N ₃ N ₄ N ₅ N ₆ ...... Ni RN _i+17 N _i+18 N _i+19 N _i+20 ...... N _n -5,

15X Xi

14X X2

13C C ₃ 10-23 DNAzyme

12 4

11

Wherein, in the schematic diagram of the binding of the 10-23 deoxyribozyme analog to the substrate, N is a nucleotide monomer composition of the substrate, and N is a nucleotide monomer composition of the 10-23 deoxyribozyme recognition domain, N is any nucleotide unit that can be complementary to WC with the purine nucleotide N. R: a purine nucleotide unit, Y is a pyrimidine nucleotide unit, and C is a nucleotide unit containing a cytosine base. The length of the 41 ^ moiety may vary with the sequence of the target nucleic acid, the composition of the target, and the number of bases of the target is m, which may be the number of gene sequences from 4 to the full length, The number of ends of the oxyribozyme analog is 11 (4-50). Xj, X ₂ , X4, X ₅ , X6, X ₈ , X ₉ , X„, X ₁₂ , X ₁₄ , X ₁₅ are introduced modified structural units.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein X" Χ ₂ , Χ ₄ , Χ ₅ , Χί, Xs, Χ ₉ , Χιι, Χΐ ₂ in the catalytic domain of the deoxyribozyme , Χΐ4, Χι _5, etc., can be similar to nucleosides as shown by structural formulas J, B, D, E, F

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein X ₂ , X6 , X _{14 are} selected for modification with guanosine analogues D and E; X ₅ , X ₉ , X„, X ₁₂ , X _1S selection was modified with adenosine analogs J and B; and Xs selection was modified with uridine analog F.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein

[In the guanidine nucleoside analogs J and D, the atom Z at the 7 position may be independently selected from carbon and nitrogen atoms. among them,

When Z is ^^, the R ^{1 in the} 7 position, R ¹ may be hydrogen, halogen (fluorine, chlorine, bromine, iodine), heterocyclic ring, aryl group and heteroaryl group (3-20), R ⁷ , or R ⁸ . In LR ⁸ , R ⁸ is a hydroxyl group, J>, a heterocyclic ring, an aromatic group and a heteroaromatic group (the number of ^^ is 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , anthracenyl group, substituted fluorenyl NH- C(NR ⁷ ₂ )=NR ⁷ , SR ⁷ , halogen, and the like. R ⁷ may be a linear fluorenyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (H0 carbon atoms), a linear and branched structure containing an aromatic ring, and the like.

L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group, or the like. In the heterocyclic ring, the aryl group and the heteroaryl group (the number of the subgroups is 3-20), one or more substituents may be taken, and the group may be R ⁹ , which has the same definition as R ⁷ .

When Z is a nitrogen atom, there is no substituent.

In the purine nucleoside analogs J, B, D, E, the substituent at the 2-position is R ² , which may be hydrogen, hydroxy, decyl, halogen, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , SR ⁷ , heterocycle, The number of aromatic and heteroaromatic groups is 3-20), R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is hydroxy, amino, heterocyclic, aryl and heteroaryl (carbon number 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl HC (NR ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, etc. L is a linker, and the linker may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having a carbon number of ≤ 10 or the like). R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having a carbon atom of <10), a linear and branched structure containing an aromatic ring, and the like. In the aryl group and the heteroaryl group (the number of broken atoms is from 3 to 20), there may be one or more substituents, which may be R ⁹ , which has the same definition as R ⁷ .

Indole nucleoside analogs In formula J and formula B, the substituent at position 6 is R ³ and may be hydrogen, J^, hydroxy, decyl, halogen, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl) ), SR ⁷ , heterocyclic ring, aryl and heteroaryl (carbon number 3-20), R ⁷ , or R ⁸ . In LR ⁸ , R ⁸ is hydroxy, ^ heterocyclic, aryl and heteroaryl (3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl HC (R ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, etc. L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having a carbon number of ≤ 10 or the like). R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a linear or branched structure containing an aromatic ring. In the case where the aryl group and the heteroaromatic group have a number of 3-20), one or more of them may be taken, and the group may be R ⁹ , which has the same definition as R ⁷ .

The purine nucleoside analogs in the formula J and the formula B may have the same or different substituents at the 2-position and the 6-position.

The purine nucleoside analogs J and D, the W at the 8-position may be a carbon atom and a nitrogen atom. among them, When W is ^, the above is taken as R ^{1 ()} , which may be hydrogen, halogen (fluorine, chlorine, bromine, ), J>, sulfhydryl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , SR ⁷ , Heterocyclic, aryl and heteroaryl, the number of 3-20), R ⁷ , or LR ⁸ . In R ⁸ , R ⁸ is a hydroxyl group, an amino group, an aromatic group and a heteroaryl group (having a carbon number of 3 to 20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , an anthracenyl group, a substituted indenyl group NH-C (NR ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, etc. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a linear or branched structure containing an aromatic ring. L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms) or the like. In the heterocyclic ring, the aryl group and the heteroaryl group (having a carbon number of 3 to 20), there may be one or more substituents, and the substituent may be R ⁹ , which has the same definition as R ⁷ .

The purine nucleoside analogs J and D have no substituent when W at the 8-position is a nitrogen atom. The purine nucleoside analogs J and D, Z and W may be the same or different.

The five-membered ring in which the purine nucleoside analogs B and E, Z and V are located is a saturated ring structure, and Z and V may be atoms such as carbon, nitrogen, oxygen, sulfur, and the like. When Z is a carbon atom, the substituent thereon is R ¹ , and when V is a carbon atom, the substituent thereon is R ¹¹ , and its definition is the same as R ¹ ; when Z is a nitrogen atom, the above is taken R ¹ , but excluding halogen and pseudohalogen; when V is a nitrogen atom, the substituent on it is R ¹¹ , which is defined the same as R ¹ but not halogen and pseudohalogen. When Z and V are oxygen or sulfur atoms, they are not taken.

The pyrimidine nucleoside analog F, the substituents R ⁴ , R ^{5 at the 5} and 6 positions, may be hydrogen, halogen (fluorine, chlorine, bromine, reef), heterocyclic ring, aryl group and heteroaryl group (number of carbon atoms) For 3-20), R ⁷ , or LR ⁸ . In the formula, R ⁸ is a hydroxyl group, an amino group, a heterocyclic ring, an aromatic group and a heteroaryl group (having a carbon number of 3 to 20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , an anthracenyl group, a substituted fluorenyl group NH-C ( NR ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, and the like. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a straight chain and a branched chain containing an aromatic ring. L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated fluorenyl group, a cycloalkyl group (having ≤ 10 carbon atoms) or the like. In the aryl group and the heteroaryl group (having a carbon number of 3-20), there may be one or more substituents, and the substituent may be R ⁹ , which is defined as the R ⁷ phase. Same.

The pyrimidine nucleoside analog F, the substituents R ⁴ and R ^{5 at the 5-} and 6-positions, may be the same or different.

The nucleoside analogs J, B, D, E, F, the sugar ring moiety may be a deoxyribose group, a six member sugar ring group, an LNA type, or other modified sugar ring structure; the sugar ring configuration may be D- or L-type.

Nucleoside analogues J, B, D, E, F, the 2,-position substituent of the five-membered sugar ring, which may be hydrogen, amino, fluorine atom, methyl group | L group, ethyl group, propyl group |曱|L-vinyl group, ethyl acetonitrile|L^, propylene, methylamino, dimethylamino, ethylamine, diethylamino, propylamine, dipropylamine, cyclopropylamine, etc.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the nucleoside analogs J, B, D, E, F are involved:

[(1) Among the purine nucleoside analogs J and D, the atom Z at the 7 position may be a carbon atom or a nitrogen atom. When Z is a carbon atom, the substituent of R ¹ at the 7 position is preferably hydrogen, fluorine, chlorine, bromine, iodine, cyano, imidazolyl, pyrazolyl, thienyl, triazolyl, pyridyl, phenyl , phenethyl, ethylphenethyl, phenylpropyl, methyl^^phenethyl, B! ^Phenylethyl, tert-butylphenethyl, or LR ⁸ . In LR ⁸ , R ⁸ is preferably hydroxy, amino, imidazolyl, fluorenyl, pyrazolyl, triazolyl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, methylphenyl , ethyl phenyl, tert-butylphenyl, naphthyl, ketone, oxime HJ ethoxy, guanylamino, dimethylamino, ethylamine, diethylamino, propylamine, dipropylamine, cyclopropylamine base.

L is preferably a linear alkyl arm of 2 to 5 carbon atoms.

LR ⁸ may be hydroxypropyl, () hydroxypropenyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methylpropyl, ethyl propyl; amine propyl, Z) amin propylene, Amine propynyl, amine butyl, amine amyl, amine hexyl, methylaminopropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-)imidazole Base, (2 or 4-) imidazolyl, (2 or 4-)imidazolium, oxime ethyl, propyl propyl, butyl butyl, pentyl, pyridine Ethyl, pyridylpropyl, phenethyl, nonylphenylethyl, ethylphenethyl, tert-butylphenethyl, anthranilylethyl, ethyl ftj^phenethyl, phenylpropyl, phenylbutyl, etc. .

When hydrazine is a nitrogen atom, there is no substituent.

(2) The purine nucleoside analog J, B, D, in the oxime, the substituent R ^{2 at the} 2-position is preferably hydrogen, J., fluorenyl, imidazolyl, hydroxy, halogen, or LR ⁸ . In LR ⁸ , R ⁸ is preferably hydroxy, amino, imidazolyl, fluorenyl, pyrazolyl, thienyl, triazolyl, pyridyl, phenyl, decylphenyl, ethylphenyl, propylphenyl, toluene Base, B! ^phenyl, tert-butylphenyl, naphthyl, anthracenyl, anthracene|L&, ethyl L&, methylamino, dimethylamino, ethylamine, diethylamino, propylamine, dipropylamine, cyclopropylamine base;

L is a linear or branched fluorenyl arm of 2 to 5 carbon atoms.

LR ⁸ may be hydroxypropyl, (£, Z) hydroxypropenyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methylpropyl, ethylpropyl; amine propyl, Z) aminpropenyl , alkynyl, aminobutyl, amine amyl, amine hexyl, methylaminopropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-)imidazole Ethyl, (2 or 4-) imidazolyl, (2 or 4-)imidazolium, oxime ethyl, propyl propyl, butyl butyl, pentyl, pyridylethyl, pyridylpropyl, phenethyl , mercaptophenethyl, ethylphenethyl, toluene ethyl, ethylphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

(3) In the purine nucleoside analogs J and D, the W of the five-membered ring is preferably a carbon and a nitrogen atom. When W is ^^ neutrons, which takes ^ ^ R ^ie may be hydrogen, halogen (fluorine, chlorine, bromine, pestle), amino, guanidino, or LR ^8. In LR ⁸ , R ⁸ is preferably hydroxy, amino, imidazolyl, fluorenyl, pyrazolyl, thienyl, triazolyl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, toluene Base, ethyl 3⁄4^phenyl, tert-butylphenyl, naphthyl, methoxy, ethyl H&, methylamino, dimethylamino, ethylamine, diethylamino, propylamine, dipropylamine , cyclopropylamine;

L is a linear or branched alkyl arm of 2 to 5 carbon atoms.

LR ⁸ may be hydroxypropyl, (E, Z) hydroxypropenyl, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, propylpropyl, ethylpropyl; amine propyl, ( , ) amine propyl Bee-based, Amine propyl group, amine butyl group, amine amyl group, amine hexyl group, guanylaminopropyl group, dimethylaminopropyl group, ethylaminopropyl group, diethylaminopropyl group; (2 or 4-) imidazole Base, (2 or 4-) imidazolyl, (2 or 4-) imidyl butyl, decyl ethyl, propyl propyl, butyl butyl, pentyl, pyridyl, pyridyl, phenethyl , mercaptophenethyl, ethylphenethyl, methyl | 1 phenethyl, ethylphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

When W is a nitrogen atom, there is no substituent.

(4) Among the purine nucleoside analogs B and E, the five-membered ring is a saturated structure, and Z and V are preferably carbon and a nitrogen atom; Z and V may be the same or different. When Z is a carbon atom, the substituent on it is R ¹ , and when V is a carbon atom, the substituent R ¹¹ on it has the same definition as R ¹ ; when Z is a nitrogen atom, the above is taken as R ¹ , but does not include halogen and pseudohalogen; when V is a nitrogen atom, the ^^ is R", which has the same definition as R ¹ but is not substituted by halogen and pseudohalogen. When Z and V are oxygen or When the bowl is atomic, there is no base.

(5) In the pyrimidine nucleoside analog F, the substituents R ⁴ and R ^{5 at the 5-} and 6-positions are preferably halogen (fluorine, chlorine, bromine, reef), imidazolyl, or LR ⁸ . In LR ⁸ , R ⁸ is preferably hydroxy, amino, imidazolyl, fluorenyl, pyrazolyl, thienyl, triaziryl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, anthracenylene , ethyl phenyl, tert-butylphenyl, naphthyl, anthracenyl, fluorene #L&, 乙, amidino, dimethylamino, ethylamine, diethylamino, propylamine, dipropylamine , cyclopropylamino; L is a linear or branched alkyl arm of 1 to 5 carbon atoms.

LR ⁸ can be hydroxypropyl, ( , ) hydroxypropenyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, propyl, B! Propyl; amine propyl, ( , ) amine propylene, amine propynyl, amine butyl, amine pentyl, amine hexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, Diethylaminopropyl; (2 or 4-)imidazolium, (2 or 4-)imidazolyl, (2 or 4-)imidazolium, oxime ethyl, propyl propyl, butyl butyl, hydrazine Pentyl, pyridylethyl, pyridylpropyl, phenethyl, methylphenethyl, ethylphenethyl, tolueneethyl, tert-butylphenethyl, ethylphenethyl, phenylpropyl, phenylbutyl Wait.

Nucleoside analogs J, B, D, E, F, the sugar ring moiety can be deoxyribose, LNA Type; The configuration of the sugar ring can be D- or L-type.

Nucleoside analogues J, B, D, E, F, 2,-position of the five-membered sugar ring, which may be hydrogen, J., sub, 曱|L&, ethylene propylene, methyl ethene, ethoxyethylene H group, propoxy vinyl fluorenyl group, etc.]. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the nucleoside analogs J, B, D, E, F are involved:

[(1) In the purine nucleoside analogs J and D, Z and W of the five-membered ring are preferably a carbon atom and a nitrogen atom; Z and W may be the same or different.

(2) In the purine nucleoside analogs J and D, the atom Z at the 7-position is independently selected from a carbon atom and a nitrogen atom. When Z ^, midnight, 7 ^, R ^ ¹ takes preferably hydrogen, fluoro, chloro, Australia, iodine, imidazole, hydroxymethyl, hydroxypropyl, (, Z) hydroxypropylene, hydroxypropyl propynyl Base, hydroxybutyl, hydroxypentyl, hydroxyhexyl, propyl, B! Propyl; amine propyl, (E, Z) amin propylene, amine propynyl, amine butyl, amine pentyl, amine hexyl, methylaminopropyl, dimethylaminopropyl, ethylaminopropyl Base, diethylaminopropyl; (2 or 4-) imidazolium, (2 or 4-) imidazolyl, (2 or 4-) imidazolium, oxime ethyl, propyl propyl, butyl butyl , pentyl, pyridylethyl, pyridylpropyl, benzyl, phenethyl, methylphenethyl, ethylphenethyl, tert-butylphenethyl, anthranilylethyl, ethylphenethyl, benzene Propyl, phenylbutyl, etc.

(3) In the purine nucleoside analogs J and D, the W of the five-membered ring is preferably a carbon and a nitrogen atom. When w is ^^ neutrons, which takes R ¹ "are each independently selected from hydrogen, fluoro, chloro, bromo, iodo, guanidino, imidazolyl, hydroxymethyl, hydroxypropyl, (C) a hydroxyl-propenyl, hydroxyethyl Propyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methylpropyl, ethylpropyl; amine propyl, (E, Z) aminpropenyl, amine propynyl, amine butyl, amine pentyl, amine hexyl, Methylaminopropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-)imidazolium, (2 or 4-)imidazolyl, (2 or 4 - )imidinyl, oxime ethyl, propyl propyl, hydrazine butyl, pentyl, pyridylethyl, pyridylpropyl, benzyl, phenethyl, nonylphenylethyl, ethylbenzene Ethyl, tert-butylphenethyl, toluene ethyl, ethylphenethyl, phenylpropyl, phenylbutyl, etc.

When W is a nitrogen atom, there is no substituent.

(4) In the purine nucleoside analogs J, B, D, E, the substituent R ^{2 at the} 2-position is preferably hydrogen, J., hydroxy, decyl, imidazolyl, halogen, hydroxyethyl, hydroxypropyl, CE , Z) hydroxypropenyl, hydroxypropyl^ & hydroxybutyl, hydroxypentyl, hydroxyhexyl, propyl propyl, propyl propyl; amine propyl, (, Z) amin propylene, amine propynyl, amine Butyl, amine pentyl, amine hexyl, methylaminopropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-) imipenyl, (2 or 4-)imidazolium, (2 or 4-)imidazolium, decylethyl, decyl propyl, hydrazinobutyl, pentyl, pyridylethyl, pyridylpropyl, phenethyl, methylphenethyl , ethyl phenethyl, anthranilyl ethyl, ethoxyphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

(5) In the purine nucleoside analogs J, B, D, E, the substituent R ^{3 at the} 6 position is preferably hydrogen, fluorenyl, hydroxy, imidazolyl, halogen, hydroxyethyl, hydroxypropyl, (,) hydroxy Propylene, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, propyl propyl, propyl propyl; amine propyl, ( , ) amine propylene, amine propynyl, amine butyl, amine pentyl , aminohexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-) sodium ethionyl, (2 or 4-) imidazolyl , (2 or 4-) butyl butyl, oxime ethyl, propyl propyl, hydrazine butyl, pentyl pentyl, pyridylethyl, pyridylpropyl, phenethyl, nonylphenylethyl, ethylphenethyl , 甲苯 ethyl, ethyl phenethyl, tert-butyl phenethyl, phenylpropyl, phenylbutyl and the like.

(6) Among the purine nucleoside analogs B and E, the five-membered ring is a saturated structure, and Z and V are preferably carbon and nitrogen atoms; and Z and V may be the same or different. When Z is ^^, it is taken as R ¹ ; when V is ^^, the ^ ^ is R", the definition is the same as R ¹ ; when Z is a nitrogen atom, R ¹ , but excluding halogen and pseudohalogen; when V is a nitrogen atom, the substituent on it is R ¹¹ , which is the same as R ¹ but not halogen and pseudohalogen. When V is an oxygen or sulfur atom, it is not taken. (7) In the pyrimidine nucleoside analog F, the substituents R ⁴ and R ^{5 at the 5-} and 6-positions are preferably halogen (fluorine, chlorine, bromine, iodine), imidazolyl, hydroxydecyl, hydroxyethyl , hydroxypropyl, ( , Z) hydroxypropenyl, hydroxypropyl; ^, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methylpropyl, ethoxypropyl; amine propyl, Z) aminpropenyl, amine Propynyl, amine butyl, amine pentyl, amine hexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl;

(2 or 4-)imidazolium, (2 or 4-)imidazolyl, (2 or 4-)imidazolium, oxime ethyl, decyl propyl, butyl butyl, pentyl, pyridylethyl, Pyridylpropyl, phenethyl, nonylphenylethyl, ethylphenethyl, A! Benzylethyl, ethylphenidyl, tert-butylphenylethyl, phenylpropyl, phenylbutyl and the like.

(8) Nucleoside analogs J, B, D, E, F, the sugar ring moiety may be a deoxyribose group, an LNA type, and the configuration of the sugar ring may be D- or L-form. The 2,-position substituent of the five-membered sugar ring may be hydrogen, 1⁄2, sub, I, I, E, E, E, E, E, E, E, E, E, E, E, E, E

The 10-23 deoxyribozyme analog according to any one of the first aspects of the invention may be used with the nucleoside analogs J, B, D, E, F for 5 dA sites X ₅ , X ₉ , X _n , X ₁₂ , X ₁₅ are modified:

(1) In the modification of 5 dA sites X ₅ , X ₉ , Xu, X ₁₂ , X ₁₅ , compounds J, B, D, E, F can be inserted into these sites instead of dA;

(2) in the modification of 5 dA sites X _s , X ₉ , X„, X ₁₂ , X ₁₅ , it is preferred to select compounds J and B instead of dA;

(3) in the modification of the five dA sites X ₅ , X ₉ , Xn, X ₁₂ , X ₁₅ , they are all positions that can be substituted;

(4) In the modification of 5 dA sites X ₅ , X ₉ , X„, X ₁₂ , X _1S , in the single substitution modification, X ₉ is a preferred modification site;

(5) In the modification of 5 dA sites X _s , X ₉ , X„, X ₁₂ , ₅ , multiple sites (2-5) can be modified at the same time.

10-23 deoxyribozyme analog according to any one of the first aspects of the invention, 4 dG The positions of Χϊ, Χ ₂ , Χβ, and ₁₄ are each independently substituted by the nucleoside analogs J, Β, D, Ε, F, which are involved, wherein:

(1) In the four dG positions, in the modification of X ₂ , Xe , X ₁₄ , compounds J, B, D, E, F can be inserted into these sites instead of dG;

(2) In the modification of 4 dG positions, Χ ₁ Χ ₂ , Χβ , X ", it is preferred to select compounds D and Ε to insert these sites instead of dG;

(3) In the four dG positions, in the modification of Χ ₂ , Χβ , Χ ₁₄ , in the case of single substitution modification, G2 and G14 are preferred sites for modification;

(4) In the modification of 4 dG positions, Χ ₂ , Χί, Xi4, multiple sites can be modified at the same time.

10-23 DNAzyme according to any one of the analogs of the first aspect of the present invention, the positions X4 and two dT X ₈ are each independently may optionally be directed to nucleoside analogs J, B, D, E, F replaced the position, where:

(1) in the modification of the position of two dTs and X ₈ , the modified monomer may be introduced simultaneously or separately;

(2) In the modification of the position of two dTs and X ₈ , compounds J, B, D, E, F can be inserted into these sites instead of dT;

(3) In the modification of the positions X4 and X ₈ of the two dTs, it is preferred to select the compound F to insert these sites instead of dT.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein the modification of the domain of the deoxyribozyme is carried out by using the above nucleoside analogs J, B, D, E, F Sites (X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ ), 4 dG modification sites (X" X ₂ , Xe , X ₁₄ ), and two dT modification sites (X4 and X) ₈ ), which can be substituted by a combination of a plurality of different types of nucleoside analogs.

The 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the single or combined substitution of the nucleoside analogs of formula J, formula 8, formula 0, formula, formula F is also associated with trona Deletion of the base (eg, deletion of T8) - modification of the catalytic domain of the 10-23 deoxyribozyme. A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the single or combined substitution of the nucleoside analog of formula J, formula 0, formula, formula F is also combined with the replacement of the natural base Modification of the catalytic domain of 10-23 deoxyribozyme.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the nucleoside analog of formula J, formula 8, formula 0, formula, formula F can also be used in the substitution of other natural monomers.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the introduction of the nucleoside analog of formula J, formula 0, formula E, formula F can be combined with the anti-nucleic acid decoration of deoxyribozyme.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, wherein the anti-nucleic acid decoration method has a thiophthalate bond skeleton; 2,-fluoro, 2,-a ftj^ V - a gas-based ethylene H (MOE), 2,-ethoxy modification, LNA, etc.; introduction of inverted nucleomonomers at 5^,-end to obtain a novel deoxyribozyme analog with higher enzyme stability .

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the nuclease-resistant modification is applicable to both the catalytic domain of the deoxyribozyme and its both end recognition domains.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the introduction of a nucleoside analog of formula J, formula 8, formula 0, formula, formula F can improve the transport of deoxyribozyme The combination of modifications performed.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein said means for improving transport comprises encapsulation of liposomes and cationic liposomes, and other transport materials; cholesterol, PEG, etc. and deoxygenation Methods such as covalent attachment of ribozymes.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the metal ion, including the divalent metal ion and the monovalent metal ion, contributes to an increase in the catalytic efficiency of the deoxyribozyme analog.

10-23 DNAzyme like according to a first aspect of the present invention, any one of, wherein said divalent metal ion is selected from _{^{^{M g 2+, Mn 2+> Pb}}} 2+, Zn 2+, Ca 2+, these Ion energy Increase the catalytic efficiency of the deoxyribozyme analog.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the divalent metal ion M _g ²⁺ is a preferred choice.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the divalent metal ion Mg ²⁺ concentration is from 0.01 to 50 mM.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the divalent metal ion Mg ²⁺ concentration is preferably 0.1-2 mMo

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the monovalent metal ion is selected from the group consisting of Na+ and K+, and the valence metal ion promotes the catalytic reaction of the deoxyribozyme analog.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the concentration of the monovalent metal ions Na+ and K+ is from 0 to 500 mM.

The 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the concentration of the monovalent metal ions Na+ and K+ is preferably 50-200 mM.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the catalytic cleavage reaction of the deoxyribozyme analog is affected by pH, and the pH ranges from 3.0 to 9.0.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein said pH is preferably from 4.0 to 9.0.

A 10-23 deoxyribonucleotide analog according to any one of the first aspects of the invention, wherein the deoxyribozyme analog has a recognition domain length of 4-25 bases each.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the sequence of the recognition domain of the two ends of the deoxyribozyme analog is designed to be complementary to the sequence fragment of any of the target nucleic acids.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the acid fragment is derived from a gene of interest to be manipulated, including a gene of interest for genetic research and gene therapy.

a 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein Deoxyribozyme analogs can be used as artificial endonucleases as molecular biology tools.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, wherein the deoxyribozyme analogs can be designed to lyse any disease-causing gene fragment as a drug candidate for gene therapy.

A 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, which is any 10-23 deoxyribozyme analog selected from the examples of the invention or which has been enumerated. The 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, for a sequence of vascular endothelial growth factor mRNA, synthesizes a 10-23 deoxyribozyme analog selected from the following numbers (Table 1): Table 1: Modification sites and modified monomers contained in the 10-23 deoxyribozyme analog

Position of monomer =3⁄4 glycoside No. Sequence composition of 10-23 deoxyribozyme analogue

Analog monomer number

DZ01 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca

Cct)-3'

LKDZ22 5'-d(tgc tct cca GGC TIG CTA CAA CGA cct gca cct)-3, A5=l

LKDZ23 5'-d(tgc tct cca GGC TAG CT1 CAA CGA cct gca A9=l

Cct)-3'

LKDZ24 5,-d(tgc tct cca GGC TAG CTA CIA CGA cct gca cct)-3' All=l

LKDZ25 5'-d(tgc tct cca GGC TAG CTA CAA CGI cct gca cct)-3, A15=l

LKDZ26 5'-d(tgc tct cca GGC TAG CTA CA1 CGA cct gca cct)-3' A12=l

LKDZ12 5'-d(tgc tct cca GGC T2G CTA CAA CGA cct gca cct)-3' A5=2

LKDZ13 5'-d(tgc tct cca GGC TAG CTA CAA CG2 cct gca cct)-3' A15=2

LKDZ14 5'-d(tgc tct cca GGC TAG CT2 CAA CGA cct gca A9=2

Cct)-3'

LKDZ15 5,-d(tgc tct cca GGC TAG CTA C2A CGA cct gca cct)-3' All=2

LKDZ16 5'-d(tgc tct cca GGC TAG CTA CA2 CGA cct gca cct)-3' A12=2 LKDZ17 5'-d(tgc tct cca GGC TAG CTA CAA CG3 cct gca cct)-3' A15=3

LKDZ18 5'-d(tgc tct cca GGC TAG CTA CA3 CGA cct gca cct 3, A12=3

LKDZ19 5'-d(tgc tct cca GGC TAG CTA C3A CGA cct gca cct)-3' All=3

LKDZ20 5'-d(tgc tct cca GGC T3G CTA CAA CGA cct gca cct)-3, A5=3

LKDZ21 5'-d(tgc tct cca GGC TAG CT3 CAA CGA cct gca A9=3 cct)-3,

LKDZ27 5'-d(tgc tct cca GGC TAG CT4 CAA CGA cct gca A9=4 cct)-3'

LKWQ01 5'-d(tgc tct cca GGC TAG CTA CAA CG6 cct gca cct)-3' A15=6

LKWQ02 5'-d(tgc tct cca GGC TAG CTA CA6 CGA cct gca cct)-3' A12=6

LKWQ03 5'-d(tgc tct cca GGC TAG CTA C6A CGA cct gca cct)-3' All=6

LKWQ04 5'-d(tgc tct cca GGC T6G CTA CAA CGA cct gca cct)-3, A5=6

LKWQ05 5'-d(tgc tct cca GGC TAG CT6 CAA CGA cct gca A9=6 cct)-3,

LKWQ06 5'-d(tgc tct cca 11GC TAG CTA CAA CGA cct gca Gl=ll cct)-3,

LKWQ07 5'-d(tgc tct cca GllC TAG CTA CAA CGA cct gca G2=ll cct)-3'

LKWQ08 5'-d(tgc tct cca GGC TAll CTA CAA CGA cct gca G6=ll cct)-3'

LKWQ09 5'-d(tgc tct cca GGC TAG CTA CAA CUA cct gca G14=ll cct)-3,

LKDZ10 5'-d(tgc tct cca GGC 21AG CTA CAA CGA cct gca T4=21 cct)-3,

LKDZ11 5,-d(tgc tct cca GGC TAG C21A CAA CGA cct gca T8 =21 cct)-3' LKDZ02 5'-d(tgc tct cca GGC 22AG CTA CAA CGA cct gca T4=22 cct)-3,

LKDZ03 5'-d(tgc tct cca GGC TAG C22A CAA CGA cct gca T8=22

Cct)-3,

LKDZ 4 5'-d(tgc tct cca GGC 23AG CTA CAA CGA cct gca T4=23

Cct)-3'

LKDZ05 5'-d(tgc tct cca GGC TAG C23A CAA CGA cct gca T8=23

Cct)-3' wherein, "the position of the modified monomer, indicating the type of monomer to be substituted and the position of the monomer, "nucleoside analog monomer number" means a nucleoside analog single selected from the following numbers Body:

) ₃ OH ) ₃ NH ₂ H ₂ C ₆ H ₅ H ₂ (4-)lm

A second aspect of the present invention provides a method for producing a 10-23 deoxyribozyme analog according to any one of the first aspects of the present invention, which comprises solid phase synthesis of 10-23 deoxygen nucleus by a conventional phosphoramidite method. Step on the analog. '

Among the modified monomers introduced, the protecting groups and deprotection methods of the respective functional groups vary depending on the type of the functional group and the nucleoside analog. For example, a protecting group for a hydroxyl group may be a tert-butylsilyl group, a tert-butylbenzene J 3⁄4, an acetyl group, a benzoyl group, a trifluoroacetyl group or the like. Protection of benzoyl, acetyl, trifluoroacetyl and the like.

For the oxime analog, the protecting group for the introduced hydroxyl group may be tert-butyldimethylsilyl, tert-butyldiphenyl 1J1⁄2, acetyl, benzoyl, trifluoroacetyl. The preferred protecting group is tert-T-dimethyl, tert-butyldiphenyl.

For pyrimidine analogs, the protecting group for the introduced hydroxyl group may be tert-butyldimethylsilyl, tert-butylbenzene, acetyl, benzoyl, trifluoroacetyl. The preferred protecting group is tert-butyl dimethyl! ^, tert-butyl diphenyl

Tert-butyl diphenyl! ^Based and tert-butyl difluorene JJ: There are two methods for deprotection of the base. The first one is concentrated ammonia water, 55-60 ° C, 18-20 hours. Another deprotection method is a 1 M solution of tetra-n-butylammonium fluoride in tetrahydrofuran, protected from light overnight at room temperature. Both methods are suitable for the case where other natural monomers are protected by common protecting groups. The second deprotection method is suitable for the case where a natural monomer protected by an easy-to-protection group is required.

The amino group introduced in the nucleoside analog may be a benzoyl group, a fluorenylmethoxycarbonyl group, an acetyl group or a trifluoroacetyl group. A preferred protecting group is a trifluoroacetyl group. This protecting group-protected monomer is suitable for use with natural monomers protected by conventional protecting groups and for use with natural monomers protected by deprotectable groups.

The separation and purification methods of the obtained 10-23 deoxyribozyme analog are reversed phase high performance liquid chromatography and denaturing polyacrylamide gel electrophoresis.

The desalting method of the obtained 10-23 deoxyribozyme analog has a gel column chromatography and a SEP-PAK column extraction.

According to the preparation method of the second aspect of the invention, the sequence of the substrate

5'- ΝΊ N'2 N'3 N' ₄ N' ₅ N' ₆ ...... RY N' _i+3 N' _i+4 N' _i+5 N' _i+6 ..·· ·· N' _m -3' The corresponding modified 10-23 deoxyribozyme analogs can be synthesized.

3'- Ni N2 N3 N4 N5 Nj+18 Ni+19 Nj+20 N _n -5'

among them,

N, which is the nucleotide monomer composition of the substrate, and N is a nucleotide monomer composition of 10-23 deoxyribozyme. R is a purine nucleotide unit, Y is a pyrimidine nucleotide unit, i is an integer of 4-33, and the substrate includes a partial sequence or a full-length sequence for genetic manipulation and gene therapy, so the m is set to a minimum of 4 (for example) n is an integer from 4 to 100; n is an integer from 4 to 50; the two-terminal recognition domain of a plurality of N-constituting 10-23 deoxyribozymes forms a Watson-Crick pair with the sequence of the substrate;

The definitions of N, n, X ₁₅ , X", X ₁₂ , X _u , X ₉ , X ₈ , X6 X ₅ , t, X ₂ , are as defined in any one of the first aspects of the invention.

The preparation method according to any one of the second aspect of the present invention, wherein the substrate may be a sequence consisting of any nucleotide monomer, including a sequence for performing genetic manipulation and gene therapy, such as a column 5 of vascular endothelial growth factor, -AGG TGC AGG AUG GAG AGC Α-3Ό A third aspect of the invention provides a kit, kit, or composition, such as a pharmaceutical composition, comprising: i) 10-23 of any of the first aspects of the invention A deoxyribozyme analog, and optionally ii) a carrier or excipient, particularly a pharmaceutically acceptable carrier or excipient, and optionally iii) product technical instructions or instructions for use. In one embodiment, the carrier or excipient is selected from the group consisting of water, sodium chloride, dextrose, mannitol, lactose, and the like.

According to a fourth aspect of the present invention, there is provided a 10-23 deoxygen nucleus according to any one of the first aspects of the present invention. Use of an enzyme analog in the preparation of a product for genetic research and/or gene therapy, such as a drug, kit, kit.

The fourth aspect of the invention also provides the use of the 10-23 deoxyribozyme analog of any one of the first aspects of the invention for the preparation of a product, such as a medicament, for use as an endonuclease or as a molecular biology tool.

The fourth aspect of the invention also provides the use of the 10-23 deoxyribozyme analog of any one of the first aspects of the invention for the preparation of a product, such as a medicament, for cleavage of any disease-causing gene fragment.

The fourth aspect of the invention also provides the use of the 10-23 deoxyribozyme analog according to any one of the first aspects of the invention for the preparation of a product, such as a medicament, for use as a drug candidate for gene therapy.

A fifth aspect of the invention provides a method of performing genetic research and/or gene therapy comprising administering to a subject in need thereof (such as, but not limited to, cells, ex vivo cells, tissues, ex vivo tissues, bacteria, viruses, microorganisms, An animal, mammal, human, etc.) is administered an effective amount of the 10-23 deoxyribozyme analog of any one of the first aspects of the invention, or the subject is as described in any one of the first aspects of the invention. -23 deoxyribozyme analog contact.

A fifth aspect of the invention also provides a method of performing an endonuclease study or a molecular biology study, comprising to a subject in need thereof (such as, but not limited to, cells, ex vivo cells, tissues, ex vivo tissues, bacteria, An effective amount of the 10-23 deoxyribozyme analog according to any one of the first aspects of the invention, or the subject and any of the first aspect of the invention, is administered by a virus, a microorganism, an animal, a mammal, a human, or the like Item 10-23 Deoxyribozyme Analog Contact - The fifth aspect of the invention also provides a method of performing cleavage of any pathogenic gene fragment comprising, to a subject in need thereof (such as, but not limited to, cells, ex vivo cells) , tissue, ex vivo tissue, bacteria, virus, microorganism, animal, mammal, human, etc.) is administered an effective amount of the 10-23 deoxyribozyme analog of any of the first aspects of the invention, or the subject is tested The body is contacted with the 10-23 deoxyribozyme analog of any of the first aspects of the invention. According to any one of the aspects of the invention, the pathogenic gene comprises or a retroproliferative gene, a virus, a genetic mutant gene or the like. Detailed description of the invention

The features and advantages of the present invention are further described below.

All documents cited in the present specification, their contents are hereby incorporated by reference, and if the meanings expressed by these documents are inconsistent with the present invention, the expression of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the ordinary meanings of those skilled in the art, and even though the present invention is intended to provide a more detailed description and explanation of the terms and phrases herein, the terms and phrases are Inconsistent with the well-known meanings, the meanings expressed by the present invention shall prevail.

As used herein, the term "animal" refers to animals such as, but not limited to, birds such as chickens, ducks, and the like, as well as mammals and the like.

As used herein, the term "mammal" refers to mammals such as, but not limited to, pigs, dogs, cats, cows, horses, and the like, as well as humans, particularly humans.

Some preferred features or further features

The present invention is structurally modified based on 10-23 deoxyribozyme, and its structure is 3,- ! N ₂ N ₃ N ₄ N ₅ N ₆ ······· NiX ₁₅ X ₁₄ C ₁₃ X ₁₂ XnC ₁₀ X ₉ X ₈ C ₇

C ₃ X ₂ Xi RN _i+17 N _i+18

N _i+19 N _i+20 ······ N _n -5,. 3,- X ₁₅ X ₁₄ C ₁₃ Xi ₂ XiiC ₁₀ X ₉ X ₈ C ₇ ^5X4

C3X2X1 -5' is a catalytic domain and is a moiety to be modified by the present invention; R is a purine base complementary to the pyrimidine base Y of the target sequence cleavage site; N represents the recognition moiety at both ends thereof, and the number of bases at both ends is the same Or different, ranging from 4 to 25. The structure of the target sequence recognized by it is 5,- ΝΊ N, ₂ N, ₃ N, ₄ ······ N'i RY NV ₃ N' _i+4 N' _i+5 N' _i+6 · ····· N, _m -3,,

R is a purine base, Y is a pyrimidine base, 5,-RY-3 is a cleavage site of a deoxyribozyme, and both ends are sequences recognized by a deoxyribozyme, which can be genetically manipulated or used for genes. A partial or full-length sequence of a disease-causing gene of treatment. Referring to Figure 2, a schematic diagram of the binding of the modified 10-23 deoxyribozyme of the present invention to a substrate, wherein N is the nucleotide monomer composition of the substrate, N is a nucleotide monomer composition of the recognition domain of the 10-23 deoxyribozyme analog, and N is any nucleotide unit that can be complementary to WC with the target nucleic acid N. R is a purine nucleotide unit, Y is a pyrimidine nucleotide unit, and the length of the mutual moiety may vary depending on factors such as the sequence of the target nucleic acid, the base composition, and the like, and the number of bases of the target is at least four, deoxyribozyme The number of identification fields at both ends is n, from 4 to 50. Xi, X ₂ , X4, X ₅ , X6, X ₈ , X ₉ , X _u , X ₁₂ , X ₁₄ , X ₁₅ are introduced modified structural units.

In the present invention, in the catalytic domain of deoxyribozyme, X ₂ , X4, X ₅ ,

X6, X ₈ , X ₉ , X„, X ₁₂ , X ₁₄ , Χ ₁₅ are chemically modified, and the nucleoside analogs involved are as shown in the following structural formulas J, B, D, E, F. The compound represented by X can

JBDEF In one embodiment of the invention, XX ₂ , X6, x _{14 is} selected for modification with guanosine analogues D and E; Χ ₅ , Χ ₉ , Χπ, Χ ₁₂ , Χ ₁₅ for adenosine analogue J and B is modified; X4 and X ₈ is modified by selecting a uridine analogue F.

In one embodiment of the invention, the definitions of the above nucleoside analogs J, B, D, E, F are as follows:

(1) Purine nucleoside analogs J and D, the atoms Z of the five-membered ring are each independently selected from a carbon atom and a nitrogen atom. among them,

When z is ^^, the R ^{1 in the} 7 position, R ¹ may be hydrogen, halogen (fluorine, chlorine, bromine, reef), heterocyclic ring, aromatic group and heteroaryl group (, the number of 3-20) , R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is a hydroxyl group, an aromatic group and a heteroaryl group (the number is 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , anthracenyl, substituted fluorenyl NH-C (NR ⁷ ₂ ) =NR ⁷ , SR ⁷ , halogen Prime, pseudohalogen, etc. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a linear or branched structure containing an aromatic ring.

L is ligated, and may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), etc., and the linking arm may also be an amide-containing bond, an ester bond, or an ether bond. , linear and branched structures of thioether bonds, and the like.

In the heterocyclic ring, the aryl group and the heteroaryl group (the number of 3-20), there may be one or more substituents, and the substituent may be R ⁹ , which has the same definition as R ⁷ .

(2) The purine nucleoside analogues J, B, D, E, the substituent at the 2-position is R ² , which may be hydrogen, hydroxy, decyl, halogen, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , SR ⁷ , The number of aromatic and heteroaromatic groups is 3-20), R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is hydroxy, amino, aryl and heteroaryl, 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (R ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, etc. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated fluorenyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a linear or branched structure containing an aromatic ring. In the aryl group and the heteroaryl group (wherein the number of 3-20), there may be one or more substituents, and the substituent may be R ⁹ , which has the same definition as R ⁷ .

(3) purine nucleoside analogs J and B, the substituent at position 6 is R ³ , which may be hydrogen, amino, hydroxy, decyl, halogen, OR ⁷ , OCOR ⁷ (ester group), NHR ⁷ , NR ⁷ ₂ , SR ⁷ , an aromatic group and a heteroaromatic group have a number of 3-20), R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is hydroxy, amino, aryl and heteroaryl (carbon number 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen, etc. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group number ≤ 10 or more, and a linear and branched structure containing an aromatic ring. In the aryl group and the heteroaryl group (having 4 to 20 carbon atoms), there may be one or more substituents, and the substituent may be R ⁹ , which has the same definition as R ⁷ .

The purine nucleoside analogs J and B may have the same or different substituents at the 2 and 6 positions.

(4) Purine nucleoside analogs J and D, which may be a ^ and a nitrogen atom. When it is ^^, it is taken as R ^1<} , which can be hydrogen, halogen (fluorine, chlorine, bromine, ). J^, fluorenyl, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , SR ⁷ , heterocyclic ring, aryl and heteroaryl (carbon number 3-20), R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is hydroxy, J^, aryl and heteroaryl (carbon number 4-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ ) = R ⁷ , SR ⁷ , halogen, etc. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a linear or branched structure containing an aromatic ring. L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), etc.; the linking arm may also be an amide-containing bond, an ester bond, an ether The bond, the linear and branched structure of the thioether bond, and the like. In the heterocyclic ring, the aryl group and the heteroaryl group (having a carbon number of 3 to 20), one or more may be taken, and it may be R ⁹ , which has the same definition as R ⁷ .

The purine nucleoside analogs J and D have no substituent when W at the 8 position is a nitrogen atom.

The purine nucleoside analogs J and D, the Z and W of the five-membered ring are preferably a carbon atom and a nitrogen atom; Z and W may be the same or different.

(5) The five-membered ring in which the purine nucleoside analogs B and E, Z and V are located is a saturated ring structure, and Z and V may be atoms such as carbon, nitrogen, oxygen, sulfur, and the like. When Z is a carbon atom, the substituent on it is R ¹ ; when V is a carbon atom, the substituent on it is R ¹¹ , which has the same definition as R ¹ ; when Z is a nitrogen atom, ^ ^ is R ¹ , but does not include halogen and pseudohalogen; when V is a nitrogen atom, the substituent on it is R ¹¹ , which is the same as R ¹ but not halogen and pseudohalogen. The bases can be the same or different. When Z and V are oxygen or sulfur atoms, there is no substituent.

(6) Pyrimidine nucleoside analog F, the substituents R ⁴ and R ^{5 at the 5-} and 6-positions, which may be hydrogen, halogen (fluorine, chlorine, bromine, iodine), heterocyclic ring, aryl group and heteroaryl group (The number is 3-20), R ⁷ , or LR ⁸ . In LR ⁸ , R ⁸ is a hydroxyl group, an amino group, a heterocyclic ring, an aromatic group and a heteroaryl group (the number of germanium atoms is 3-20), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , anthracenyl group, substituted fluorenyl group NH- C(NR ⁷ ₂ )=NR ⁷ , SR ⁷ , halogen, and the like. R ⁷ may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having ≤ 10 carbon atoms), and a straight chain and a branched chain containing an aromatic ring. Structure, etc. L is a linking arm, and the linking arm may be a linear alkyl group, a branched alkyl group, an unsaturated alkyl group, a cycloalkyl group (having a carbon number of ≤ 10 or the like). In the aryl group and the heteroaryl group (having a carbon number of 3 to 20), there may be one or more substituents, and the substituent may be R ⁹ , which has the same definition as R ⁷ .

(7) nucleoside analogs J, B, D, E, F, the sugar ring portion thereof may be deoxyribose, other five member glycosyl groups, six member sugar ring groups, LNA type, or other modified sugar ring structure The configuration of the sugar ring can be D- or L-form.

In one embodiment of the invention, the nucleoside analogs J, B, D, E, F, the 2,-position substituent of the five member sugar ring, may be hydrogen, ^, sub, 曱|L&, Ethylpropoxy, methyl IL-ethylene H-based, ethylidene E-based, propylethylene H-based, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamine, Cyclopropylamine and the like.

In one embodiment of the present invention, in the purine nucleoside analogs J and D, the Z atom at the 7 position is preferably a carbon atom and a nitrogen atom, and when Z is a carbon atom, the R ¹ at the 7 position is taken. Preferred are hydrogen, fluorine, chlorine, bromine, iodine, *J>, imidazolyl, pyrazolyl, pyridinium, phenyl, tolyl, ethylphenyl, propylphenyl, nonyloxyphenyl, ethoxy Phenyl, naphthyl, anthracenyl, or LR ⁸ . In LR ⁸ , R ⁸ is preferably hydroxy, imidazolyl, fluorenyl, pyrazolyl, triazolyl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, nonyloxyphenyl, B ! ^phenyl, naphthyl, anthracenyl, anthracene, ethyl L &, amidino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, cyclopropylamino;

L is preferably a linear or branched alkyl arm of 1 to 5 carbon atoms;

LR ⁸ may be hydroxypropyl, (, Z) hydroxypropenyl, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methyl propyl, ethyl propyl; amine propyl, ( , Z) Aminopropenyl, amine propynyl, amine butyl, amine amyl, amine hexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2- or 4-) imidazolium, (2- or 4-)imidazole Base, (2- or 4-), oxazolidine, decylethyl, decyl propyl, butyl butyl, pentyl, pyridylethyl, pyridylpropyl, phenethyl, methylphenethyl, Phenylethyl, anthranilylethyl, ethylphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

In one embodiment of the invention, in the purine nucleoside analogs J, B, D, E, the R ^{2 in} position ² is preferably hydrogen, hydroxy, imidazolyl, fluorenyl, halo, or LR ⁸ . In L- ⁸ R, R ^{8 is} preferably hydroxyl, amino, imidazolyl, guanidino, pyrazolyl, pyrazolyl group, phenyl, tolyl, ethylphenyl, propylphenyl, Yue phenyl, ethyl benzene ί Base, tert-butylphenyl, naphthyl, anthracenyl, methyl iL&, ethyl, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, cyclopropylamino; a linear or branched alkyl arm of 1-5 carbon atoms;

LR ⁸ may be hydroxypropyl, Z) hydroxypropenyl, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, methylpropyl, ethyl fL^propyl; amine propyl, (, Z) amin propylene Base, amine propynyl, amine butyl, amine pentyl, amine hexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2- or 4- Imidazolylethyl, (2- or 4-)imidazolium, (2- or 4-)imidazolium, oxime ethyl, propyl propyl, butyl butyl, pentyl, pyridylethyl, propyl , phenethyl, methyl phenethyl, ethyl phenethyl, oxime ethyl, ethyl phenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

In one embodiment of the invention, in the purine nucleoside analogs J and D, the W of the five member ring is preferably a carbon and nitrogen atom. When W is a ^, it is preferably R ¹ "which may be hydrogen, fluoro (chloro, chloro, bromo, iodo), ^ fluorenyl, imidazolyl, or LR ^8. In LR ⁸ , R ⁸ is preferably a hydroxy group. , imidazolyl, fluorenyl, pyrazolyl, thienyl, triazolyl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, nonyloxyphenyl, ethylphenyl, tert-butyl Phenyl, naphthyl, anthracenyl, methyl, ethyl | L&, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, cyclopropylamino;

L is a linear or branched alkyl arm of 2-5.

LR ⁸ may be hydroxypropyl, (JE, Z) hydroxypropenyl, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, decyl, ethyl propyl; amine propyl, ( , ) amine propylene base, Aminopropynyl, amine butyl, amine amyl, amine hexyl, methylaminopropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2 or 4-)imidazole Base, (2 or 4-) imazethyl, (2 or 4-) imidenyl, decyl, decyl, hydrazino, pentyl, pyridyl, pyridyl, phenyl Base, methylphenethyl, ethylphenethyl, methyl IUL phenethyl, ethylphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl and the like.

When W is a nitrogen atom, there is no substituent.

In one embodiment of the present invention, in the purine nucleoside analogs J and D, Z and W of the five-membered ring are preferably carbon and nitrogen atoms, and Z and W may be the same or different.

In one embodiment of the invention, in the purine nucleoside analogs B and E, the five member ring is a saturated structure, and Z and V are preferably carbon, nitrogen atoms; and Z and V may be the same or different. When Z is a carbon atom, the substituent on it is R ¹ ; when V is a carbon atom, the substituent on it is R ¹¹ , which has the same definition as R ¹ ; when Z is a nitrogen atom, the substitution thereon The base is R ¹ , but does not include halogen and pseudohalogen; when V is a nitrogen atom, it is taken as R ¹¹ , and its definition is the same as R ¹ , but does not include halogen and pseudohalogen. When Z and V are oxygen or bowl atoms, there is no substituent.

In one embodiment of the present invention, the pyrimidine nucleoside analogs F in the 5-position and 6 taken RJ ^ R ^4, R ⁵ is preferably a halogen (fluorine, chlorine, bromine, iodine), imidazolyl, or LR ⁸ . In LR ⁸ , R ⁸ is preferably hydroxy, imidazolyl, fluorenyl, pyrazolyl, pyridyl, phenyl, tolyl, ethylphenyl, propylphenyl, decylphenyl, ethylphenyl, tert-butylbenzene , naphthyl, anthracenyl, aiLi, ethyl L, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, cyclopropylamino; L is 1-5 carbon atoms Linear or branched alkyl arm;

LR ⁸ may be hydroxypropyl, ()hydroxypropenyl, hydroxypropynyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, decylpropyl, ethylpropyl; amine propyl, (, Z) aminpropenyl, Amine propynyl, amine butyl, amine amyl, amine hexyl, guanylpropyl, dimethylaminopropyl, ethylaminopropyl, diethylaminopropyl; (2- or 4-)imidazole Ethyl, (2- or 4-)imidazolyl, (2- or 4-)imidazolium, oxime ethyl, propyl propyl, butyl butyl, pentyl, pyridine Ethyl, pyridylpropyl, phenethyl, nonylphenylethyl, ethylphenethyl, oxime phenethyl, ethylphenethyl, tert-butylphenethyl, phenylpropyl, phenylbutyl, etc. .

In one embodiment of the invention, in the nucleoside analogs J, B, D, E, F, the sugar ring moiety can; an oligoribose group, a six member sugar ring group, an LNA type, or other modified sugar ring structure The configuration of the sugar ring can be D- or L-type.

In one embodiment of the invention, the nucleoside analogs J, B, D, E, F, the 2,-position substituent of the five member sugar ring, may be hydrogen, Κ^, fluorine atom, methoxy Base, B

3⁄4^, C, A|1 base ethyl, ethoxyethyl propyl ethoxy ftj methylamino, diammonium, ethylamine, diethylamino, propylamine, dipropylamine, cyclopropylamine Base _beta

In one embodiment of the invention, the invention utilizes the above nucleoside analogs J, B, D, E, F to modify the 10-23 deoxyribozyme to obtain novel deoxyribozyme analogs.

In one embodiment of the present invention, the present invention utilizes the above nucleoside analogs J, B, D, E, F to "decorate" five dA sites X ₅ , X ₉ , X _n , X ₁₂ , X _1S , The invention may take one or more of the following more specific embodiments:

(1) In the modification of 5 dA sites Χ ₅ , Χ ₉ , Χ _π , Χ , 2 , Χ ₁₅ , they are all positions that can be replaced;

(2) In the modification of 5 dA sites Χ ₅ , Χ ₉ , Χ „ , Χ ₁₂ , Χ ₁₅ , in the single substitution modification, Χ ₉ is the preferred modification site;

(3) In the modification of 5 dA sites Χ ₅ , Χ ₉ , Χ „, Χ ₁₂ , Χ ₁₅ , multiple sites (2-5) can be modified at the same time;

(4) In the modification of 5 dA sites Χ ₅ , Χ ₉ , Χ „, Χ ₁₂ , Χ ₁₅ , nucleoside analogs “1, B, D, E, F can be inserted into these sites instead of dA ;

(5) In the modification of the five dA sites X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ , it is preferred to select one or more of the nucleoside analogs J and B instead of the dA site.

In one embodiment of the invention, the invention utilizes the above nucleoside analogs J, B, D, E, F for the four dG positions XX ₂ in the catalytic domain of the 10-23 deoxyribozyme, Χί, X ₁₄ is modified, and X _P Χ ₂ , Χβ, Χ ₁₄ are all positions that can be substituted at the four dG positions of the deoxyribozyme. The invention may employ one or more of the following more specific embodiments:

(1) In the position of 4 dG, in the modification of X" X ₂ , X6, X ₁₄ , in the case of single substitution modification, the sites modified with G2 and G14 are preferred;

(2) In the modification of X d 的₂ , Χ , Χ ₁₄ at four dG positions, multiple sites can be modified at the same time;

(3) In the modification of X d, X ₂ , X6 , X ₁₄ , nucleoside analogues J, B, D, E, F can be inserted into these sites instead of dG;

(4) In the modification of 4 dG positions, Χ ₁₅ Χ ₂ , Χβ, Χ ₁₄ , it is preferred to select compounds D and Ε to insert these sites instead of dG.

In one embodiment of the present invention, the present invention utilizes the above nucleoside analogs J, B, D, E, F of the two dT catalytic domain of the 10-23 DNAzyme position X4 and X ₈ be modified , at two positions X4 dT and the DNAzyme X ₈ are used to modify the site. The invention may take one or more of the following more specific embodiments:

In one embodiment of the present invention, the present invention utilizes the above nucleoside analogs J, B, D, E, F to modify the catalytic domain of 10-23 deoxyribozyme, 5 dA modification sites (Χ ₅ , Χ ₉ , Χπ, Χ ₁₂ , Χ ₁₅ ), four dG modification sites (X" X ₂ , Xe, Xi4), and two dT modification sites (X4 and X ₈ ), each independently Optionally, it is replaced by a combination of one or more different types of nucleoside analogs J, B, D, E, F.

In one embodiment of the invention, the above nucleoside analogs B, D> E, F Single or combination substitutions can also be modified with deletions of natural bases (eg, deletion of T8)

Catalytic domain of 10-23 deoxyribozyme.

In one embodiment of the invention, the single or combined substitution of the above nucleoside analogs B, D, E, F may also modify the catalytic domain of the 10-23 deoxyribozyme along with the replacement of the natural base.

In one embodiment of the invention, single or combined substitutions of the above nucleoside analogs B, D, E, F may also be used for the substitution of other natural monomers.

In one embodiment of the invention, the single or combined substitution of the above nucleoside analogs J, B, D, E, F can be combined with modifications that increase the stability of the deoxyribozyme analog. The method for nuclease-resistant modification is a thionate linkage skeleton; 2,-fluoro, 2,-methoxy, V-methoxyvinyloxy (MOE), 2,-ethylidene, 2,- Ethoxyethyleneoxy modification, LNA, etc.; and introduction of inverted nucleomonomers at the 3'-end to obtain novel 10-23 deoxyribozyme analogs with higher enzyme stability.

In one embodiment of the present invention, wherein the stability of the deoxyribozyme is improved, both the catalytic domain of the deoxyribozyme and the recognition domain at both ends thereof can be used.

In one embodiment of the invention, the introduction of a nucleoside analog of formula J, formula 8, formula 0, formula, formula F can be combined with modifications to improve the transport of deoxyribozymes.

In one embodiment of the invention, the means for improving transport includes liposomes and cationic liposomes, and encapsulation of other transport materials; covalent attachment of cholesterol, PEG, etc. to deoxyribozymes, and the like. In one embodiment of the present invention, for the two-end recognition sequence of the 10-23 deoxyribozyme analog, respectively, 1^ to 1^, and 111 ₊₁₇ to 1^ are respectively indicated, and N constitutes the recognition portion of both ends thereof, and The monomer complementary to the target sequence, R is a purine base, which is complementary to Y of the target sequence cleavage site; the number of Chengji at both ends is the same or different, ranging from 4 to 20.

The 10-23 deoxyribozyme analog of the present invention can be directed to any RNA sequence, To design complementary sequences at both ends.

Sequence fragments to which the present invention is directed include genes for structural and functional studies, as well as stem genes for gene therapy. Evaluation of the activity of novel 10-23 deoxyribozyme analogs

The activity of the cleavage substrate of the deoxyribozyme analog and the prototype deoxyribozyme of the present invention is tested under certain conditions, including the composition and concentration of the buffer, the concentration of the deoxyribozyme analog, the pH, and the divalent metal. The type and concentration of ions.

The composition of the buffer is related to the range of pH. Different buffers are selected depending on the pH. Such as Na-MES (6.0, 6.5), Na-HEPES (pH 7.0), Tris-HCl (pH 7.5, 8.0, 8.5). Existing work has shown that these counterions have no effect on catalysis.

In the present invention, the catalytic reaction of these deoxyribozyme analogs is affected by pH, pH from 3.0 to 9.0, or 6.0-9,0, with a suitable range of 7.0-7.5, and preferably close to physiological 7.5.

In the present invention, the catalytic reaction of these deoxyribozyme analogs requires the participation of ions. In particular, the participation of divalent metal ions, including M _g ²⁺ , Mn ²⁺ , Zn ²⁺ , Fe ²⁺ , Pb ²⁺ , Ca ^{2+ ,} etc. can all be promoted.

Among the divalent metal ions, Mg ²⁺ is the best choice. Concentrations range from 0.01 mM to 100 mM. The range of 0.1-2 mM close to the intracellular Mg ²⁺ concentration is suitable, and 0.1-0.5 mM Mg ²⁺ is the optimal concentration range.

The catalytic reaction of monovalent ions to these deoxyribozyme analogs also accelerates. Monovalent ions are generally K+, Na ⁺ , at a concentration of 5-500 mM, with an optimum concentration of 50-200 mM.

The catalytic reaction of these deoxyribozyme analogs is influenced by the concentration of the enzyme and substrate. Therefore, the catalytic reaction efficiency is measured under conditions of single conversion and multiple conversion. The concentration ratio of enzyme to substrate is from 10:1 to 1000:1 under a single conversion condition. The concentration ratio of enzyme to substrate is 1:10-1:1000 under multiple conversion conditions. The concentration of the enzyme ranges from 10 μΜ to 0.1 ηΜ, and the concentration of the substrate ranges from 100 μΜ to 0.1 ηΜ. Study on the catalytic mechanism of novel deoxyribozyme analogues

In the present invention, a deoxyribozyme having a higher catalytic efficiency than the prototype 10-23 deoxyribozyme was obtained, and a new deoxyribozyme analog > s was 11 times as large as the prototype 10-23 deoxyribozyme. Therefore, the present invention has studied its catalytic mechanism.

The present invention studies the effects of various factors on the catalytic efficiency from several factors affecting catalysis. The deoxyribozyme analog and the prototype deoxyribozyme were compared under the same conditions.

The catalytic reaction kinetics of the deoxyribozyme analogs were determined under both conditions of single conversion and multiple conversion. Under a single conversion condition, the substrate to enzyme concentration ratio is from 10:1 to 1000:1, and under multiple conversion conditions, the concentration ratio of the two is from 1:10 to 1:1000. The concentration of the enzyme ranges from 0.1 ηΜ to 100 μΜ, and the concentration of the substrate ranges from 0.1 ηΜ to 100 μΜ.

The effect of ρΗ on the catalytic rate: different compositions with different buffers. The buffer used was from ρΗ3-9.0.

Effect of metal ion species during the reaction were to evaluate the effects of divalent ions, including _{^{^{M g 2+, Mn 2+, Zn}}} 2+, Fe 2+, Pb 2+, Ca 2+ , etc. on the reaction.

Comparing the effects of the above various factors on the modified and prototype deoxyribozymes, the inventors believe that the chemical reaction mechanism of the modified deoxyribozyme analog of the present invention is the same as that of the prototype. The increase in the catalytic rate of the former may be due to its better catalytic reaction conformation. DRAWINGS

Figure 1 is a schematic diagram of a complex of 10-23 deoxyribozyme and a nucleic acid substrate, wherein R is a fluorene monomer, Y is a pyrimidine monomer, N is a nucleomonomer of a nuclear species, and m is a N; N is the nucleotide monomer at both ends of the deoxyribozyme, n is its number, and is matched with the N of the nucleic acid substrate by Watson-Crick; the arrow indicates the location where the nuclear is cleaved.

Figure 2 is a schematic representation of the binding of a modified 10-23 deoxyribozyme analog to a substrate. In the figure, N is the nucleotide monomer composition of the substrate, and N is the nucleotide sequence of the 10-23 deoxyribozyme recognition domain. The body composition, N is any nucleotide unit that can be paired with the target nucleic acid N and can be WC complementary.

R is a purine nucleotide unit, Y is a pyrimidine nucleotide unit, and the length of each of the 13⁄4 portions may vary depending on factors such as the sequence of the target nucleic acid, the composition of the minus group, and the substrate may be a part for performing genetic manipulation or gene therapy. The sequence or full length sequence may be at least 4; the number of ends of the deoxyribozyme is n (4-50). X _n X ₂ , X4, X ₅ , Xs, X ₉ , Xu, Xn, X ₁₄ , X ₁₅ are introduced modified structural units.

Figures 3 and 9 show the results of a cleavage reaction between each modified deoxyribozyme analog and unmodified 10-23 deoxyribozyme DZ01, respectively. Detailed ways

The invention is further illustrated by the following examples and examples, but it should be understood that these examples and experimental examples are only used in more detail and are not to be construed as limiting this invention.

The present invention provides a general and/or specific description of the materials used in the tests as well as the test methods. While many of the materials and methods of operation used to accomplish the objectives of the present invention are well known in the art, the present invention is still described in detail herein. It will be apparent to those skilled in the art that, hereinafter, the materials and methods of operation of the present invention are well known in the art unless otherwise specified. Example 1: Design of Partial Nucleoside Analog Monomers for Design of Deoxyribozyme Modified Nucleoside Analogs See compounds 1-24 below (also known as nucleoside analogs 1-24):

1 : R ¹ = H 6: R, = H 11 : R, = H

2: R ¹ = (CH ₂ ) ₃ OH 7: R ¹ = (CH ₂ ) ₃ OH 12: R ¹ = (CH ₂ ) ₃ OH 3: R ¹ = (CH ₂ ) ₃ NH ₂ 8: R1 = ( CH ₂ ) ₃ NH ₂ 13: R ¹ = (CH ₂ ) ₃ NH ₂ 4: R ¹ = CH ₂ CH ₂ C ₆ H ₅ 9: R ¹ = CH ₂ CH ₂ C ₆ H ₅ 14: R ¹ = CH ₂ CH ₂ C ₆ H ₅ 5: R ¹ = CH ₂ CH ₂ (4-) lm 10: R ¹ = CH ₂ CH ₂ (4-) lm 15: R ¹ = CH ₂ CH ₂ (4-) lm

16: R ¹ = H 21 R = CH ₂ OH

17: R ¹ = (CH ₂ ) ₃ OH 22 R ⁴ = CH ₂ CH ₂ OH

18: R ¹ = (CH ₂ ) ₃ NH ₂ 23 R ⁴ = CH ₂ CH ₂ CH ₂ OH

19: R ¹ = CH ₂ CH ₂ C ₆ H ₅ 24 R = CH ₂ CH ₂ (4-) lm

20: R ¹ = CH ₂ CH ₂ (4-)lm

In the above formulas, Ι^=<:Η ₂ <:Η ₂ (4-)Ιιη indicates that the R ¹ group is 2-(4-imidazolyl)-ethyl-, and has similar meanings for the R ⁴ group. .

Referring to the above formulas, in the design of nucleoside analogs, for purine nucleoside analogs, their five-membered rings are predominant, as shown by compounds 1 and 11, their positions of N7 and C8 are exchanged, and further introducing at the C7 position of the arm an alkyl (e.g., C _r C ₆ alkyl arm, for example arm alkyl dC ₄₎ attached J ^, a hydroxyl group, an imidazolyl group, and phenethyl group, to give 2-deoxy-like fragrance glands -5 and deoxyguanosine analogues 12-15.

In the five-membered ring modification of the purine nucleoside analog, N7 is substituted with a carbon atom to give compounds 6 and 16. Further introducing a functional group attached at the 7 position with an alkyl arm (for example, a C _r C ₆垸 group arm, for example, a dC ₄ alkyl arm), to obtain an analog 7-10 and a deoxyguanosine analog 17-20 .

In the pyrimidine nucleoside analog, the 5-position modification of the pyrimidine nucleoside is dominant, such as a compound. 21-24, a hydroxyl group, a phenyl group, and an imidazolyl group attached to an alkyl arm (e.g., a dC ₆ alkyl arm such as a dC alkyl arm) are introduced at the five positions, respectively. Example 2: Synthesis of nucleoside analogs

The chemical synthesis of known nucleoside analogs, and the corresponding phosphoramidite monomers required for their introduction into nucleic acid sequences, can be synthesized according to the skill of the art or by methods disclosed in the literature, new nucleosides The analogs and their corresponding phosphoramidite monomers can be synthesized by our laboratory design.

In the present invention, purine nucleoside analogs 2-5, 7-10, 12-15, and 17-20 may be coupled with 7- 7-deaza-8-nitro-deoxyadenosine using a monosubstituted ethynyl group. The reaction and subsequent catalytic hydrogenation are obtained.

The hydroxy-containing pyrimidine compound 21-23 was obtained by a literature method using a total synthesis method.

23 and 24 can be obtained by coupling between 5-carboside and 3-hydroxypropene block or imidazole acetylene, and subsequent catalytic hydrogenation. Example 3: Synthesis of a phosphoramidite monomer of a nucleoside analog The functional group introduced by an orthogonal protection strategy suitable for DNA solid phase synthesis requires the introduction of a hydroxyl group in the synthesis of a phosphoramidite monomer. For orthogonal protection, the selected protecting group must be suitable for solid phase synthesis by DNA phosphoramidite method.

The compounds of the following formula I to formula VI show partial deoxyadenosine analogs of the invention

IV V VI

The compound of the following formula vn to formula XII shows the structure of the phosphoramidite monomer of the partial deoxyguanosine analog of the present invention:

X XI XII The compound of the following formula XIII to formula XVI shows the phosphoramidite monomer structure of the partial pyrimidine nucleoside analog to which the present invention is applied:

XIII XIV XV NC

XVI

In the present invention, the synthesis of the monomers represented by the formulae I, V, VII, X, XIII, XIV and XV can be referred to the literature.

In the present invention, in the phosphoramidite monomer of the purine nucleoside analog, the hydroxy protecting group at the 7 position of the formulae II and VIII may be an acetyl group, a benzoyl group, a silyl group such as a tert-butyldimethyl group and a tert-butyl group. Phenylalkyl and the like. Tert-butyldimethylalkyl and tert-butyldiphenylalkyl are preferred and the like.

In the phosphoramidite monomer in the purine nucleoside analogs, and of formula IX, 7 ΠΙ amino protecting group may be acetate, benzene Yue acyl, trifluoroacetyl and the like, to select the preferred trifluoroacetyl _β

In the phosphoramidite monomer of the purine nucleoside analog, the imidazolyl groups of formula VI and XII may be unprotected.

In the phosphoramidite monomer of the purine nucleoside analog, the 6-J^ of the base of formula I-VI can be protected with a benzoyl group or an easily deprotectable group such as di(n-butyl)aminodecenyl; The 2-K of the VII-XII is protected with isobutyryl.

The 5,-hydroxyl group of all monomers can be protected with 4,4,-dimethoxytrityl (DMT). For the phosphoramidite monomer 嘧啶ιπ-χνι of pyrimidine compounds, their hydroxyl group at the 5-position can be protected by tert-butyldiphenylsilyl; the imidazole group at the 5-position of XVI can be left unprotected. 5. The hydroxyl group can be protected by DMT. Example 4: Synthesis of phosphoramidite monomer II

The synthetic route design of 7-(3-hydroxypropyl)-7-deaza-8-nitro-deoxyadenosine 2 and its phosphoramidite monomer II is illustrated as follows:

NCCH ₂ CH ₂ 0' ^N ( ^iPr ) 2

II

(i) 3-tert-butyldiphenylphosphonium silicon oxy-oxygen propyl block

(3-tert-butyldiphenylsiloxypropyne), Pd (PPh ₃ ) ₄ , CuI, Et ₃ N, in DMF, room temperature, 8 h;

(ii) H ₂ , Pd/C, 5 atm., room temperature;

(iii) 1 M TBAF in THF at room temperature for 10 min;

(iv) (C ₄ H ₉ ) ₂ NCH(OCH ₃ ) ₂ in MeOH for 2 h;

(v) DMTC1 in pyridine, room temperature;

(vi) (CCH ₂ CH ₂ 0)(IPr) ₂ NPCl, (iPr) ₂ EtN, in CH ₂ C1 ₂ , room temperature,

30 min.

The hydroxypropyl introduction of this nucleoside analog is introduced by means of hydroxypropyne and 7- 7-deaza-8-nitro-deoxyadenosine, as shown in the above scheme. Hydroxypropyne hydroxy! It was previously protected by tert-butyldiphenylsilyl (TBDPS) (2a) to obtain orthogonal protection between this primary hydroxyl group and the 5,-hydroxyl group of the deoxyribose moiety. 2a is subjected to catalytic hydrogenation to give intermediate 2b, which is deprotected with 1 M TBAF/THF to give nucleoside analog 2. 2b of 6-JJ3⁄4 bis(n-butyl) 曱 ^ ^ [dwelling (intermediate 2c), the 5,-hydroxy group is protected with DMT (intermediate 2d), and then subjected to a phosphorylation reaction to obtain a monomer II suitable for solid phase synthesis of DNA.

Step 1.1 1-(2-de-τβ-D-erythroside) -3-(3-tert-butyldiphenyl propyl)pyrazine J3, 4-dl-pyrimidine-4-amine (2a).

7-Iodo-8-aza-7-deaza-2,-deoxyadenosine (0.38 g, 1.0 mmol) was dissolved in dry DMF (5-50 ml), and a catalytic amount of Pd was added to the solution [ P(C ₆ H ₅ ) ₃ 】 ₄ and Cul. Nitrogen was bubbled through and 3-tert-butyldiphenylsiloxypropyne (0.57 g, 2.0 mmol) and triethylamine (1-10 ml,) were added at 0-50. C reaction for 5-20 hours. Purified by column chromatography to give the product as a colorless solid (0.45g, 84%) TLC. . (CH 2 Cl 2 / CH 3 OH 9: 1) R f 0.58 UV (MeOH):

(DMSO-i ₆ ): δ 1.0 (s, 9 H, tert- u), 2.29 (m, 1 H, C2'-H„), 2.80 (m, 1 H, C2,-Hp), 3.56, 3.41 (2 m, 2 H, C5,-H), 3.85 (m, 1 H, C4'-H), 4.47 (m, 1 H, C3'-H), 4.73 (s, 2 H, 5-C≡ C 3⁄4), 4.81 (t, =5.8, 1 H, C5,-OH), 5.32 (d, /=4.4, 1 H, C3'-OH), 6.58 (t, =6.4, 1H, Cl'-H ), 7.72-7.44 (2m, 10 H, arom. H), 8.26 (s, 1 H, C2-H). Analytically calculated C ₂₉ H ₃₃ N ₅ 0 ₄ Si. (M 543.69): C, 64.06; H, 6.12; N, 12.88. Found C, 63.56; H, 6.06; N 12.76.

Step 1.2 l-(2-de-erythro-ribose)-3-(3-tert-butyldiphenyloxypropane)^--111-pyridyl ¹¹ έί3, 4-dl-pyrimidine-4-amine (2b).

In a stainless steel reactor, compound 2a (2.9 g, 3.07 mmol) was dissolved in methanol (20-200 ml) and 10% Pd/C (0.1-2 g), sealed, and hydrogen was introduced (5-15 k pressure). ), and stir for 5-10 hours at room temperature. When the reaction was stopped, nitrogen was bubbled through and filtered, and the filtrate was concentrated to give a colorless solid (2.7 g, 90.3%), Rf (CH ₂ Cl ₂ /CH ₃ OH 9:1 ) 0.53. UV (MeOH) : λ^ _χ 207 (48 400), 259 (8300), 278 (8200). ^ MRiDMSO-^): δ 0.98 (s, 9 H, tBu), 1.94 (m, 2 H, 5-CH ₂ C^ ₂ CH ₂ OH), 2.16 (m, 1 H, C2'-H _a ), 2.73 (m, 1 H, C2, -H _p ), 3.02 (m, 2 H, 5-C^ ₂ CH ₂ CH ₂ OH), 3.36, 3.51 (2 m, 2 H, C5'-H), 3.76 (m, 3 H C4, -H, 5-CH ₂ CH ₂ CiT ₂ OH), 4.41 (m, 1 H, C3' -H), 4.81 (t, =6.4, 1 H, C5,-OH), 5.22 (d, 7=4.5, 1 H, C3'-OH), 6.49 (t, /=6.3, 1 H, Cl'-H), 7.10-7.70 (2 m, 12 H, Arom. H, H ₂ ), 8.16 (s, 1 H, C2-H). Analytically calculated C ₂₉ H ₃₇ N ₅ 0 ₄ Si. 1/2H ₂ 0 (M 556.69), C, 62.51; H, 6.83 ; N, 12.57. Found C, 62.72; H, 6.77; N, 12.28.

Step 1.3 1-(2-^- -β-Ρ-erythro r-nuclear-3-(3-tert-butyldiphenoxypropanyl-1)-4 (di-n-butyl)aminoalkenyl -Amino-1H-pyrazole i3, 4-dl-pyrimidine Addition of V, TV-di-n-butylcarbamoyl diacetal acetal (0.58 g, 2.82 mmol) to a solution of compound 2b (1.50 g, 2.81 mmol) in methanol After the reaction mixture was stirred at 20-60 ° C for 4-20 hours, it was concentrated under reduced pressure and the product was purified by column chromatography. The product was obtained as a colorless oily liquid (1.70 g, 90%), R _f (CH ₂ Cl ₂ /CH ₃ OH 15:1) 0.57. UV (MeOH): ^ 214 (21 600), 319 (21 500). ¹ H MR (DMSO-rf ₆ ): 60.83, 0.90 (2 t, 6 H, N =CHN(CH ₂ CH ₂ CH ₂ <3⁄4) ₂ 】" 0.96 (s, 9 H, tBu), 1.26 (m, 4 H, N=CHN(CH ₂ CH ₂ C3⁄4CH ₃ ) ₂ ], 1.56 (m, 4 H,

N=CHN(CH ₂ C^ ₂ CH ₂ CH ₃ ) ₂ ], 2.06 (m, 2 H, 5-CH ₂ C3⁄4CH ₂ OH), 2.19 (m, 1 H, C2'-Ha), 2.77 (m, 1 H, C2,-H _p ), 3.07 (m, 2 H, 5-C^ ₂ CH ₂ CH ₂ OH), 3.37, 3.49 [2 m, 6 H, N=CHN (C^2CH ₂ CH ₂ CH ₃ ) ₂ , C5,-H], 3.72 (t, J=6.4, 2 H, 5-CH ₂ CH ₂ 3⁄4OH), 3.81 (m, 1 H, C4'-H), 4.43 (m, 1 H, C3,-H), 4.80 (t, /=5.9, 1 H, C5'-OH), 5.25 (d, /=4.5, 1 H, 3'-OH), 6.53 (t, /=6.6, 1 H , Cl'-H), 7.34-7.58 (m, 10 H, arom. H), 8.42 (s, 1 H, C2-H), 8.97 [s, 1 H, N=CTM(CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ 】. Analytically calculated C ₃₈ H ₅₄ N ₆ 0 ₄ Si, l/4H ₂ 0 (M 691.46): C, 65.94; H, 7.88; N, 12.15. Found C, 65.65; 8.19; N, 11.94.

Step 1.4 l-『2H 5-0-(4, 4,-Dimethylbenzenemethyl)-D-Red

-ribose 1-3-(3-tert-butyldiphenyloxypropionate 〖(di-n-T)-methylenylamino-lH-pyrazole B, 4-dl-pyrimidine (2d).

Compound 2c (1.20 g, 1.78 mmol) was dissolved in pyridine (2-10 ml) at 20-50. C stir Mix and add DMT-C1 (0.76 g, 2.23 mmol). After completion, the product was isolated as a colorless solid (1.06 g, 61%). R _f (CH ₂ Cl ₂ /CH ₃ OH 20:1) 0.45. UV (MeOH):

205 (77 300), 318 (30 400). ¹ HNMR (DMSO-rf ₆ ): δ 0.80, 0.91 (t, 6 H, N=CH (CH ₂ CH ₂ CH ₂ CH3) ₂ ), 0.95 (m, 9 H, tBu), 1.20 (m, 4 H, N=CHN(CH ₂ CH ₂ C3⁄4CH ₃ ) ₂ ), 1.55 (m, 4 H, N=CHN(CH ₂ C3⁄4CH ₂ CH ₃ ) ₂ ), 1.83 ( m, 2 H, 5-CH ₂ C^2CH ₂ OH), 2.27 (m, 1 H, C2'-H„), 2.71 (m, 1 H, C2, -H _p ), 3.00 (m, 4 H , C5,-H, 5-C^ ₂ CH ₂ CH ₂ OH), 3.47 [m, 4 H, N=CHN(CT ₂ CH ₂ CH ₂ CH ₃ ) ₂ ], 3.64 (m, 8 H, 20CH ₃ , 5-CH ₂ CH ₂ C r ₂ OH), 3.95 (m, 1 H, C4'-H), 4.50 (m, 1 H, C3,-H), 5.28 (d, /=5.0, 1 H, C3'-OH), 6.58-7.58 (m, 24 H, arom. H, Cr-H), 8.44 (s, 1 H, C2-H), 8.98 (s, 1 H, N=CTM (CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ). calcd for C ₅₉ H ₇₂ N ₆ 0 ₆ Si (M 989.33): C, 71.63; H, 7.34; N, 8.49. Found C, 71.31; H, 7.50; , 8.30.

Step 1.5 l-n-de^5-0-(4, 4,-dimethylbenzene, -β-D-erythr

-ribose 1-3-(3-tert-butyldiphenyl M oxypropyl^-ΐ) 〖(二正ΤΜ.) methylal-1-amino-1 Η-pyrazole [3, 4-dl and pyrimidine 3-ίί2 -cyanoethyl) hydrazine, hydrazine-diisopropylaminophosphoramidite 1 (H)'

Compound 2d (0.85 g, 0.87 mmol) was dissolved in dry dichloromethane (10-100 ml), diisopropylethylamine (1 ml, 5.75 mmol) and diiso-amino-bri 0.25 g, 1.0 mmol). The mixture is between 20 and 50. C is stirred for 1-5 hours. The mixture was diluted with dichloromethane, was washed with 5% NaHC0 ₃ solution and saturated brine, dried without sodium 7j ¾; ^, concentrated and column chromatography the product was isolated as a colorless solid (0.35 g, 35.4%). R _f (CH ₂ CI ₂ /CH ₃ OH 30:1) 0.61, 0.71. ^J H NMR (CDCI ₃ ): δ θ.86, 0.98 (t, 6 H, N=CHN(CH ₂ CH ₂ CH ₂ C^ 3) ₂ ], 1.01 (m, 9 H, tBu), 1.14-1.40 (m, 10 H, N(CH(CT ₃ ) ₂ ,

1.60 (m, 4 H, N=CHN(CH ₂ CT ₂ CH ₂ CH ₃ ) ₂ ], 1.94-2.17 (m, 3 H, 5-CH ₂ CT ₂ CH ₂ OH, C2'-H„), 2.36 -2.63 (m, 3 H, 5-0^ ₂ <:3⁄40: 011, C2'-H _p ), 2.92-3.91 (m, 18 H, C5,-H, 5-CH ₂ CH ₂ C3⁄4OH, N=CHN(C ₂ CH ₂ CH ₂ CH ₃ ) ₂ , 20C ₃ , OC^ ₂ CT ₂ CN], 4.23 (m, 1 H, C4 ,-H), 4.80 (m, 1 H, C3'-H), 6.66-7.64 (m, 24H, arom. H, Cl'-H), 8.48 (s, 1 H, C2-H), 8.86 ( s, 1 H, N=C»N(C^ ₂ CH ₂ CH ₂ CH ₃ ) ₂ 】. ³¹ P NMR (CDC1 ₃ ): 148.75, 148.49. Example 5: Nucleoside analogue 3 and its phosphorous Synthesis of amide monomer m

synthesis.

III

As shown in the above scheme, the introduction of the 3-aminopropyl group of this nucleoside analog is by the coupling reaction of 3-aminopropyne with 7- 7-deaza-8-nitro-deoxyadenosine and subsequent hydrogenation reaction. Introduced. The 3-* propyne ^* is protected by a trifluoroacetyl group prior to introduction. After the 6-position of this compound is protected by di-n-butylamine formyl group, DMT is introduced at the 5,-position, followed by phosphorylation at 3,-OH to obtain monomer III for deoxyribozyme similarity. Synthesis of matter.

1) 1-(2-Deτβ-D-ribofuranosyl)-3-(3-trifluoroacetamidopropyl) 4H-pyrene And i3,4-dl pyrimidine- 4- 3b,.

Compound 3a ((2 g, 5 mmol ) and Pd / C (10%, 1.0 g) in methanol (20_ ² 00 ml) were mixed in a stainless steel reactor, introducing hydrogen (1-5 kg), sealed and stirred at 30.C 5 hours the reaction mixture was filtered and the filtrate was concentrated to give a colorless solid (1.9 g, 95%) R f (C¾Cl 2 / CH 3 OH 10: 1).. 0.45 ^ -NMR (DMSO. -i ₆ ): δ 1.87 (m, 2 H, 5-CH ₂ C^ ₂ CH ₂ ), 2.20 (m, 1 H, C2, -H _a ), 2.79 (m, 1 H, C2'-H _p ), 2.97 (m, 2 H, 5-C^ ₂ CH ₂ CH ₂ ), 3.30 (m, 2 H, 5-CH ₂ CH ₂ C^2), 3.37, 3.52 (2 m, C5'-H) , 3.79 (m, 1 H, C4'-H), 4.42 (m, 1 H, C3'-H), 4.78 (m, 1 H, C5'-OH), 5.22 (d, = 4.76, 1 H, C3'-OH), 6.50 (t, / = 6.58, 1 H, Cl'-H), 7.35 (br, 2 H, NH ₂ ), 8.16 (s, 1 H, C2-H), 9.41 (m, 1 H, NHCO). ¹³ C NMR (DMSO-i/ ₆ ): δ 25.2, 27.3, 37.9, 62.5, 71.2, 83.7, 87.5, 98.8, 111.6, 114.5, 117.4, 120.2, 145.0, 154.8, 155.9, 158.1.

2) l-(2-de-irB-D-ribofuranosyl)-3-ethylpropyl)~4-, N-di-n-butylamino, methoxyl-1·Λ}-4Η-pyrazole 3,4-dI pyrimidine (3d

Compound 3b (1.9 g, 4.7 mmol) was dissolved in methanol (20-100 ml), and N,N-di-n-butylaminocarbazide acetal (1.75 ml, 7 mmol) was added. The solution was stirred for 1-5 hours at 20-60 ° C, then, this solution was concentrated to give the product as a colorless solid (2.1 g, 82%) purified by column _{_{_{chromatography. R f (CH 2 Cl 2}}} / CH 3 OH 20:1) 0.35. ^NMR (DMSO-i ₆ ): δθ.92 (2 t, / = 7.4, 6 H, N=CHN(CH ₂ CH ₂ CH ₂ 3⁄4) ₂ ], 1.32 (m, 4 H , N=CHN(CH ₂ CH ₂ (3⁄4CH ₃ ) ₂ ], 1.61 (m, 4 H,

N=CHN(CH ₂ CH2CH ₂ CH ₃ ) ₂ ], 2.00 (m, 2 H, 5-CH ₂ C«2CH ₂ ), 2.24 (m, 1 H, C2,-H _a ), 2.82 (m, 1 H, C2, -H _p ), 3.03 (m, 2 H, 5- 3⁄4CH ₂ CH ₂ ), 3.23-3.60 [m, 8 H, N=CHN(C^T ₂ CH ₂ CH ₂ CH ₃ ) ₂ , C5'-H, 5-CH ₂ CH ₂ C^ ₂ ] 3.82 (m, 1 H, C4'-H), 4.45 (m, 1 H, C3'-H), 4.77 (t, J = 5.7, 1 H, C5,-OH), 5.24 (d, J = 4.5, 1 H, 3'-OH), 6.55 (t, J = 6.4, 1 H, Cl'-H), 8.42 (s, 1 H, C2 -H), 8.98 [s, 1 H, N=CTM(CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ], 9.41 (m, 1 H, NHCO). ¹³ C NMR (DMSO-rf ₆ ): δ 13.5 , 13.6, 19,1, 19.8, 25.7, 27.3, 28.7, 30.4, 37.9, 45.4, 51.4, 62.5, 71.2, 83.8, 87.5, 106.0, 111.6, 114.5, 117.4, 120.2, 146.5, 155.4, 157.2, 162.5. Analytical calculation of the following formula: C ₂₄ H ₃₆ F ₃ N ₇ 0 ₄ (M 543.58): C, 53.03; H, 6.68; N, 18.04. Found: C,

52.65; H, 6.49; N, 17.87.

3) li(2-desfuran nucleus-5-(4,4,-dimethylbenzoquinone)1-3-(3-trifluoroacetamidopropyl)-4-, Ν-di-n-butyl Amino, alkenyl 1 · ί-4Η-pyrazole and 3,4-d, pyrimidine (3d)

Compound 3c (1 g, 1.85 mmol) was dissolved in dry pyridine ( 1-5 ml), EtOAc (EtOAc) Then, the reaction mixture was concentrated, the product was isolated by column chromatography, as a colorless solid (0.95 g, 62.5%) R f (CH 2 Cl 2 / CH 3 OH 20: 1). 0.7 Χ ΗΝΜΚ (DMSO-rf. ₆ ): δ 0.93 [t, 6 H, N=CHN(CH ₂ CH ₂ CH ₂ C3⁄4) ₂ ], 1.33 (m, 4 H,

N=CHN(CH ₂ CH ₂ C ₂ CH ₃ ) ₂ ), 1.62 (m, 4 H,

N=CHN(CH ₂ C3⁄4CH ₂ CH ₃ ) ₂ ), 1.77 (m, 2 H, 5-CH ₂ C^ ₂ CH ₂ ), 2.30 (m, 1 H, C2'-H„), 2.78 (m, 1 H, C2,-H _p ), 2.89 (m, 2 H, 5-CH ₂ Cn ₂ CU ₂ ), 3.03-3.13 [m, 4 H, N=CHN(C3⁄4CH ₂ CH ₂ CH ₃ ) ₂ ], 3.50-3.60 (m, C5'-H, 5-CH ₂ CH ₂ C3⁄4), 3.67, 3.69 (2 s, 2 OCH ₃ ), 3.95 (m, 1 H, C4,-H), 4.52 (m, 1 H, C3'-H), 5.27 (m, 1 H, 3'-OH), 6.60 (m, 1 H, Cl'-H), 6.73-7.30 (m, arom. H), 8.44 (s, 1 H, C2-H), 9.00 [s, 1 H, N=CTM(CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ], 9.37 (m, 1 H, NHCO). ¹³ C Wake R (DMSO-i ₆ ): δ 13.5, 13.6, 19.1, 19.8, 25.7, 27.2, 28.6, 30.4, 38.2, 45.4, 51.4, 54.8, 54.8, 64.9, 71.3, 83.4, 85.1, 85.4, 106.0, 112.8, 112.9, 114.5, 117.4, 126.3 , 127.5, 127.6, 129.5, 129.6, 135.6, 145.0, 146.5, 155.4, 157.1, 157.8, 157.9, 162.4. Analytical calculations for the following formula: C ₄₅ H ₅₄ F ₃ N ₇ 0 ₆ 0.25 Η ₂ 0 (Μ 850.45) : C, 63.55; Η, 6.46; Ν, 11.53. Found: C, 63.32; Η, 6.20; Ν, 11.40.

4) 1-ί(2-desfurose ribose) -5-(4, 4 dimethyl phenylmethyl) 1-3- (3- Trifluoroethylaminopropyl) 4-{[(N,N-di-n-butylamino)methenyl 1 ^Μ -4Η-pyrazole

Compound 3d (0.45 g, 0.55 mmol) was dissolved in dry dichloromethane (5-20 ml), then diisopropylamine tetrazolium salt (0.17 g, 1.1 mmol) and 2-cyanoethyldiisopropyl Giophosphamide (0.55 g, 1.1 mmol). This mixture was stirred at room temperature for about 2 hours. It was then washed with a 5% NaHC0 ₃ 7J solution and brine. The organic layer was dried over anhydrous Na ₂ S0 _4, concentrated and purified by column chromatography method, to give the product as a colorless solid (0.45 g, 80.5%) R f (CH 2 Cl 2 / CH 3 OH 40: 1). 0.48 , 0.50. ^MR (DMSO-i ₆ ): δ 0.90-1.81 [m, 28 H, N=CHN(CH ₂ C# ₂ C# ₂ 3⁄4) ₂ , 5-CH ₂ C^ ₂ CH ₂ ), NCH ( 3⁄4 ₂ ], 2.47-3.18 [m, 10 H, C2'-H, 5-C^ ₂ CH ₂ CH ₂ ), N=CHN(C CH ₂ CH ₂ CH ₃ ) ₂ , CH ₂ C3⁄4CN], 3.35- 3.77 [m, 14 H, C5,-H,

C ₂ CH ₂ CN], 4.10 (m, 1 H, C4'-H), 4.78 (m, 1 H, C3'-H), 6.61-6.75, 7.14, 7.28 (3 m, 14 H, Cl,- H, arom. H), 8.47 (2s, 1 H, C2-H), 9.01 [s, 1 H, N=CH (CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ], 9.40 (m, 1 H, NHCO ³¹ P NMR (CDC1 ₃ ): 147.16, 147.83. Example 6: Synthesis of nucleoside analog 4 and its phosphorous oligomonomer IV

Synthesis of the body.

4a 4b NCCH ₂ CH ₂ 0' ^N ( ^iPr >2

IV

(i) H ₂ , Pd/C, room temperature, 3 hr;

(ii) (C ₄ H ₉ ) ₂ NCH(OCH ₃ ) ₂ in MeOH, 2 h;

(iii) DMTC1 in pyridine, room temperature, 1 h;

(iv) (NCCH ₂ CH ₂ 0)(IPr) ₂ NPCl, (iPr) ₂ EtN, in CH ₂ C1 ₂ , room temperature, 30 min.

As shown in the above scheme, the introduction of the phenethyl group of this nucleoside analog is introduced by the hydrogenation reaction of phenylacetylene with 7- 7-deaza-8-nitro-deoxyadenosine. After the 6-position is protected by di-n-butylamine decanoyl group, DMT is introduced at the 5,-position, followed by phosphoramidation at 3'-OH to obtain monomer IV for deoxyribozyme analog. synthesis.

1) 1-(2-de-uranose,-3-phenyl^-4H-pyrazolo-3-d, pyrimidine-4-(4a)

7-Break 7-deaza-8-aza-deoxyadenosine (0.38 g, 1 mmol) was placed in a dry reaction flask and dissolved in dry DMF (5-50 ml), stirred under nitrogen, and phenylacetylene (0.8- 4 ml), stirring, adding triethylamine (0.2-5 ml), the solution turned black purple and stirred at room temperature for 6-30 hours. After the reaction was completed by TLC, the solvent was evaporated under reduced pressure and purified by column chromatography to afford product 0.29 g, yield: 81.2%, Rf (CH ₂ Cl ₂ /CH ₃ OH 9:1) 0.48. ^NMR^-DMSO): δ 8.27(s, 1H, H-2), 7.75, 7.50 (2m, 5H, arom. H), 6.58 (t, 1H, l'H, J=6Hz), 5.30(d , 1H, 3,-OH, J=4.8Hz), 4.79(t, 1H, 5,-OH, J=6Hz), 4.44(m, 1H, 3'H), 3.82(m, 1H, 4,- H), 2.39 (2m, 2H, 5, -H), 2.81 (m, 1H, 2, -H _p ), 2.28 (m, 1H, 2, -H _a ).

Elemental analysis: C ₁₈ H ₁₇ N ₅ 0 ₃ (Mol. Wt. 351.36) Theoretical values: C 61.53, H 4.88, N 19.93;

Found: C 61.43, H 4.88, N 19.97.

2) H2-de-τβ-D-ribofuranosyl 3-phenethyl-1H-pyridinium 3,4-d]pyrimidine-4- Compound 4a (1.8 g, 5.06 mmol) was dissolved in anhydrous methanol (50) In -300 ml), 10% Pd/C (ll-3.2 g) was added, and after reacting in an autoclave for 4-10 hours, the reaction was confirmed by TLC. The catalyst was filtered off, the solvent was evaporated, solid product was recrystallized to give 1.7 g, yield _{93.4%, Rf (CH 2 CI} 2 / CH 3 OH 9: 1) 0.38. ^MR^-DMSO): δ 8·2( s, 1Η, 2-H), 7.27(m, 5H, arom. H), 6.50(t, 1H, 1,-H, J=6.5Hz), 5.22 (d, 1H, 3'-OH, J=4.2Hz), 4.79(t, 1H, 5,-OH, J=5.7Hz), 4.43(d, 1H, 3'-H, J=4.2Hz), 3.80(m, 1H, 4,-H), 3.31(2m, 4H, 5,-H, CH ₂ ), 3.01(m, 2H, CH ₂ ), 2.74(m, 1H, 2,-Hp), 2.19 (m, 1H, 2, -H„).

Elemental analysis: C ₄₈ H _S2 N ₆ 0 ₅ ( ol. Wt. 355.39)

Theoretical values: C 60.83, H 5.96, N 19.71;

Found: C 60.56, H 5.99, N 19.22.

3) l-(2-de-τβ-D- < ribose,-3-phenylethyl-4-Gu, Ν-di-n-butylamino, mesyl 1 }-1Η-pyrazole 〖3, 4 -d〗 Pyrimidine (4b)

Synthesis with intermediate 2c. Compound 13 (1.0 g, 2.85 mmol) and synthetic N,N-di-n-butylformyl dimethyl benzoate (0.58 g, 2.85 mmol) in methanol (4 - 40 ml) at 20-60 ° C after stirring the reaction column chromatography to obtain a colorless oil 1.13 g, yield _{94.0%, Rf (CH 2 Cl} 2 / CH 3 OH 15: 1) 0.57. Rf (CH ₂ Cl ₂ /CH ₃ OH 9:1) 0.64, Rf (CH ₂ Cl ₂ /CH ₃ OH 20:1) 0.25. ¹ H NMR (< _{6 -} DMSO): S 8.98 (s, 1H, N = CH), 8.42 (s, 1H, 2-H), 7.24 (m, 5H, arom. H), 6.54 (t, 1H, l '-H, J=6.4Hz), 5.25(d, 1H, 3,-OH, J=4.48Hz), 4.81(t, 1H, 5,-OU, J=5.45Hz), 4.43(m, 1H, 3'-H), 3.80(m, 1H, 4'-H), 3.48, 3.33 [2m, 8H, 5,-H, N(CH ₂ ) ₂ , CH ₂ ]; 3.09(m, 2H, CH ₂ ), 2.78(m, 1H, 2,-H _p ), 2.22(m, 1H, 2,-H _tt ), 1.56(m, 4H, CH ₂ , CH ₂ ), 1.29, 1.16(2m, 4H, CH ₂ , CH ₂ ), 0.91, 0.67 (2t, 6H, 2CH ₃ ).

Elemental analysis: C ₂₇ H ₃₈ N ₆ O ₃ .0.5H ₂ O (Mol. Wt. 499.13)

Theoretical value: C 64.91, H 7.71, N 16.83;

Found: C 65.08, H 7.97, N 16.33.

4) li(2-desfuran ribose) -5-(4,4,-dimethoxytriphenylhydrazine 1-3-phenylethyl-4-, Ν-di-n-butylamine-based alkenyl-1) Synthesis of -1Η-pyrazolo-3,4-d-pyrimidine (4c) with intermediate 2d. Compound 4b (0.87 g, 2.07 mmol) and DMT-C1 (0.78 g, 2.28 mmol) in dry pyridine (1- Reaction in 10 ml). Column chromatography yielded 1.1 g of a colorless solid, yield 66.7%, Rf (CH ₂ Cl ₂ /CH ₃ OH 15:1) 0.57 , Rf (CH ₂ Cl ₂ /CH ₃ OH 20:1) 0.45. ^ MR^^-DMSO): δ 9.01(s, 1H, N=CH), 8.47(s, 1H, 2-H), 7.18~6.63(m, 19 H, arom. H, l'-H), 5.31(d, 1 H, 3'-OH, J=4.76Hz), 4.81(t, 1 H, 5,-OH, J=5.45Hz), 4.54(m, 1 H, 3 '-H), 3.98(m, 1 H, 4,-H), 3.63(2s, 6 H, 20CH ₃ ), 3.48[m, 4 H, N(CH ₂ ) ₂ ], 3.13(m, 4 H , 5,-H, CH ₂ ), 2.75(m, 3 H, 2'-H _p , CH ₂ ), 2.33(m, 1 H, 2,-H _a ), 1.56(m, 4 H, CH ₂ , CH ₂ ), 1.30, 1.10 (2m, 4 H, CH ₂ , CH ₂ ), 0.91, 0.74 (2t, 6 H, 2CH ₃ ).

Elemental analysis: C ₄₈ H ₅₆ N ₆ 0 ₅ . (Mol. Wt. 797.00)

Theoretical values: C 72.34, H 7.08, N 10.54;

Found: C 72.04, H 7.08, N 10.27.

5) 1-ί 2-^ -β-Ρ-ribofuranose,-5-(4,4,-dimethoxytriphenylmethyl, 1-3-phenylethynyl-4-display, Ν-二正Butyryl, methylal-1amino i-lH-pyrazole i3, 4-dl pyrimidine 3,-"f2-cyanoethyl"-N,N-diisopropylphosphoramidite 1 (IV)

Synthesis of the same phosphoramidite monomer Π. Compound 4c (0.25 g, 031 mmol) and DIEA (1 - 5 ml) and smudge reagent (0.1 - 0.5 g) were reacted in dichloromethane (20 - 50 ml). Obtained by column chromatography as a colorless solid foam 120mg, yield _{38.3%, Rf (CH 2 Cl} 2 / CH 3 OH 30: 1) 0.61, 0.67. NMR (CDC1 ₃ ): 68.88 ( s, 1H, N = CH), 8.49 ( s, 1H, 2-H), 7.28 - 6.66 (m, 19H, arom. H, 1 Ή), 4.83 (m, 1 H, 3'H), 4.23( m, 1 H, 4, H), 3·82~3·24( m, 16 H, 20CH ₃ , N(CH ₂ ) ₂ , 5Ή, OCH ₂ CH ₂ C), 3.1~2.4( m, 6H, 2, H _p , CH ₂ , CH ₂ , 2, H„,), 1.63~1.07( m, d, 22 H, 2 CH ₂ -CH ₂ , 2 CH(CH ₃ ) ₂ 0.95, 0.80 ( 2t, 6 H, 2CH ₃ ) ³¹ P NMR (CDC1 ₃ ): 148.75, 148.57. Example 7: Obtaining a 10-23 deoxyribozyme analog using a nucleoside analog

A chimeric sequence of 5,-d (agg tgc agg) AU was synthesized using a column of vascular endothelial growth factor mRNA (5,-AGG TGC AGG AU GGA GAG C Α-3', 19 1⁄2) as a substrate. - d (gga gag ca)-3', as a stem for screening highly active deoxyribozymes, wherein 5,-AU-3, is an RNA monomer, is a cleavage site.

For this chimeric 耙 sequence, the corresponding 10-23 deoxyribozyme analog was synthesized, as shown in Table 2-4, and the recognition portion (lower case letter) at both ends was a complementary fragment.

The corresponding natural nucleosides in the catalytic domain of the 10-23 deoxyribozyme are replaced by nucleoside analogs, respectively. Deoxyadenosine analogs 1-3 (the structures of which are respectively referred to in Example 1, respectively) are substituted for A5, A9, All, A12, or A15, respectively; and A9 is substituted with 4. The deoxyguanosine analog 11 replaces Gl, G2, G6, or G14, respectively; and the deoxyuridine analog 21-23, which replaces T4 or T8, respectively.

The purine nucleoside analog 1, substituting A5, A9, All, A15, A12 of 10-23 deoxyribozyme DZ01, respectively, gave novel deoxyribozyme analogs LKDZ22, LKDZ23, LKDZ24, LKDZ25, and LKDZ26 of the present invention.

The purine nucleoside analog 2, which substitutes A5, A15, A9, All, A12 of 10-23 deoxyribozyme DZ01, respectively, gives the novel deoxyribozyme analogs LKDZ12, LKDZ13, LKDZ14, LKDZ15, and LKDZ16 of the present invention.

The purine nucleoside analog 3, which replaces A15, A12, All, A5, A9 of 10-23 deoxyribozyme DZ01, respectively, gives the novel deoxyribozyme analogs LKDZ17, LKDZ18, LKDZ19, LKDZ20, and LKDZ21 of the present invention.

a purine nucleoside analog 4, substituting A9 of 10-23 deoxyribozyme DZ01, to obtain the present invention The novel deoxyribozyme analog LKDZ27.

The purine nucleoside analog 6, replacing A15, A12, All, A5, A9 of 10-23 deoxyribozyme DZ01, respectively, gave the novel deoxyribozyme analogs LKWQ01, LKWQ02, LKWQ03, LKWQ04, and LKWQ05 of the present invention.

The purine nucleoside analog 11, which replaces Gl, G2, G6, G14 of 10-23 deoxyribozyme DZ01, respectively, gives the novel deoxyribozyme analog LKWQ06, LKWQ07 of the present invention,

The pyrimidine nucleoside analog 21, which substitutes T4 or T8 of 10-23 deoxyribozyme DZ01, respectively, yields the novel deoxyribozyme analogs LKDZ10 and LKDZ11 of the present invention.

The pyrimidine nucleoside analog 22, which replaces T4 or T8 of 10-23 deoxyribozyme DZ01, respectively, yields the novel deoxyribozyme analogs LKDZ02 and LKDZ03 of the present invention.

The pyrimidine nucleoside analog 23, which replaces T4 or T8 of 10-23 deoxyribozyme DZ01, respectively, gives the novel deoxyribozyme analogs LKDZ04 and LKDZ05 of the present invention.

In the present invention, LKDZ and LKWQ and the subsequent Arabic numerals are representative symbols for indicating the 10-23 deoxyribozyme analog of the present invention, and the corresponding sequences correspond to the context of the present invention, as listed in Table 2 below. Sequence structure.

Table 2 10-23 deoxyribozyme analogs containing nucleoside analogues 1, 2, 3, 4 and 6

Table 3 10-23 deoxyribozyme analogs containing nucleoside analog 11

Example 8: Synthesis and purification method of 10-23 deoxyribozyme analog

A substrate with vascular endothelial growth factor, 5,-AGG TGC AGG AU GGA GAG CA-3', but with the corresponding chimeric sequence as the substrate, ie, 5'-d (AGG TGC) AGG)-rAU-d(GGA GAG CA)-3,, the cleavage site is an RNA residue, and the recognition part is a DNA structure. It is purchased from Dalian Bao Biotech Co., Ltd., and all other deoxyribozyme sequences are synthesized by themselves, in 392 DNA. The synthesizer was synthesized by the phosphoramidite method. The phosphoramidite monomer of the natural deoxynucleoside was purchased from Proligo, and the deoxyribozyme analogs of Tables 2-4 were synthesized together with the modified monomers. All sequences were separated and purified by denaturing gel electrophoresis, desalted, and time-of-flight mass spectrometry to determine molecular weight (Table 5). In the deprotection method of the deoxyribozyme analog sequence, when there is a hydroxyl group-containing modified monomer, concentrated ammonia water is used at 55. C incubation temperature 16-20 hours; or use 1 M TBAF/THF at room temperature, avoid light, incubate for 24 hours.

In the deprotection method of the hydroxyl group, both deprotection methods are suitable for protection from the common protective monomer; the latter is also suitable for combination with the deprotection of the easily removable monomer.

In the deprotection method of the deoxyribozyme analog sequence, when there is a modified monomer containing an amino group, concentrated ammonia is used at 55. C Incubate for 16-20 hours, or incubate for 4 hours at room temperature.

The two deprotection methods of the amino group are suitable for both the protection of the ordinary monomer and the protection of the easy-protection monomer.

Table 5. Molecular Weight of 10-23 Deoxyribozyme Analogs Determined by MALDI-TOF

Example 9: Radiolabeling method for substrate

The substrate sequence is 5,-d (agg tgc agg) -rAU-d (gga gag ca)-3, labeled with ³² P at its 5,-end. Example 10: Comparison of the catalytic ability of each deoxyribozyme analog with 10-23 deoxyribozyme was evaluated under single conversion conditions to screen for a more active 10-23 deoxyribozyme analog.

³² P-labeled 5'-d (agg tgc agg) -rAU-d (gga gag ca)-3,, 0.1 nmol, enzyme 10 nmol, target to enzyme ratio 1:100, at 50 mM Tris HC1, It was carried out in a reaction system of 50 mM Mg ²⁺ and pH 8.0. The stop solution contained 8 M Urea and 100 mM EDTA (analytical grade). 5ul were taken at 0, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, and an equal volume of stop solution was added. The reaction results were analyzed by a 20% polyacrylamide gel, and the results were shown by a phosphor screen self-developing method.

The purine nucleoside analog 1 (see the structure of Example 1, the same below), respectively, substituted A5, A9, All, A15, A12 of 10-23 deoxyribozyme DZ01 to obtain deoxyribozyme analogs LKDZ22, LKDZ23, LKDZ24, LKDZ25, and LKDZ26. The catalytic rate is: LKDZ25 « LKDZ22, LKDZ24 < DZ01 < LKDZ26 « LKDZ23.

The purine nucleoside analog 2, which replaces A5, A15, A9, All, A12 of 10-23 deoxyribozyme DZ01, respectively, gives deoxyribozyme analogs LKDZ12, LKDZ13, LKDZ14, LKDZ15, and LKDZ16. The catalytic rate is: LKDZ12 < LKDZ13 < LKDZ15 < LKDZ16 < DZ01 « LKDZ14.

The purine nucleoside analog 3, which replaces A15, A12, All, A5, A9 of 10-23 deoxyribozyme DZ01, respectively, gives deoxyribozyme analogs LKDZ17, LKDZ18, LKDZ19, LKDZ20, and LKDZ21. The catalytic rate is: LKDZ20 < LKDZ17 < LKDZ19 < LKDZ18 < DZ01« LKDZ21.

Purine nucleoside analog 4, substituted for A9 of 10-23 deoxyribozyme DZ01, deoxygenated The ribozyme analog LKDZ27, which is more active than DZ01.

In the 9th position, the dA «t adenosine analog 1-4 was substituted, and all of the 10-23 deoxyribozyme analogs were obtained with higher catalytic activity.

In the present invention, a modification site for a high rate of catalysis is found by modification of 10-23 deoxyribozyme.

The purine nucleoside analog 11 replaces Gl, G2, G6, and G14 of 10-23 deoxyribozyme DZ01, respectively, to obtain deoxyribozyme analogues LKWQ06, LKWQ07, LKWQ08, LKWQ09, and the catalytic rate is LKWQ08 « LKWQ06 < LKWQ07 -

In the present invention, by modifying the 10-23 deoxyribozyme, a second modification site which enhances its catalytic efficiency is found.

The pyrimidine nucleoside analog 21, which replaces T4 or T8 of 10-23 deoxyribozyme DZ01, respectively, obtains LKDZ10 and LKDZll. The catalytic rate of LKDZ10 is slower than that of DZ01, and the catalytic rate of LKDZ11 is comparable to that of DZ01. Note Τ4 is a more conservative 1⁄2, and Τ8 is not a conservative one.

The pyrimidine nucleoside analog 22, which replaces Τ4 or Τ8 of 10-23 deoxyribozyme DZ01, respectively, obtained LKDZ02 and LKDZ03. The catalytic rate of LKDZ02 was slower than that of DZ01, and the catalytic rate of LKDZ03 was comparable to that of DZ01. Note Τ4 is more conservative, and Τ8 is not a conservative one.

The pyrimidine nucleoside analog 23, which replaces Τ4 or Τ8 of 10-23 deoxyribozyme DZ01, respectively, obtained LKDZ04 and LKDZ05. The catalytic rate of LKDZ04 was slower than that of DZ01, and the catalytic rate of LKDZ05 was comparable to that of DZ01. Note Τ4 is more conservative, while Τ8 is not a conservative one.

The two dT residues of 10-23 deoxyribozyme have different effects on the catalytic activity. When the pyrimidine nucleoside analog 21-23 replaces the T4 of the 10-23 deoxyribozyme, the introduction of 21 has the least effect on the deoxyribozyme, which is comparable to the prototype deoxyribozyme DZ01. The catalytic rates of 22 and 23 both nucleases were reduced. When the pyrimidine nucleoside analog 21-23 replaces the T8 of 10-23 deoxyribozyme, it has little effect on the deoxyribozyme, and its catalytic ability is comparable to that of the prototype deoxyribozyme DZ01. Example 11: Study on the mechanism of high efficiency deoxyribozyme LKDZ21

In the present invention, highly efficient deoxyribozymes LKDZ14, LKDZ21, LKDZ23, LKDZ27, LKWQ09 and the like are obtained by introduction of deoxyadenosine analogs.

Taking LKDZ21 as an example, the catalytic mechanism of 10-23 deoxyribozyme analogues was evaluated based on the relationship between catalysis and efficiency.

The activity was evaluated by LKDZ21 under physiological conditions.

The catalytic rate of LKDZ21 under single conversion conditions was evaluated. The higher the concentration of the enzyme, the faster the shear rate. Its catalytic rate is much higher than the prototype DZ01.

The catalytic rate of LKDZ21 was evaluated under multiple conversion conditions. It has a higher catalytic rate than DZ01.

At near physiological concentrations of M _g ²⁺ is the most important evaluation thus respectively containing 2 mM, 0.2 mM, 0.1 mM Mg ^2+, and of 50 mM Tris-HCl (pH7.5) a ^, the evaluation of the catalytic LKDZ21 active. Its catalytic activity is higher than DZ01.

The effect of pH on the shear rate of deoxyribozyme was investigated under buffer conditions of 2 mM Mg ²⁺ and 50 mM Tris-HCl.

At pH values of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, the shear rates of LKDZ21 and DZ01 increased with increasing pH, which was positively correlated with the enhanced nucleophilic capacity of 2,-OH.

The effects of other divalent metal ions on the catalytic rates of Mn ²⁺ , Ca ²⁺ , Zn ²⁺ , deoxyribozymes LKDZ21 and DZ01 indicate that they have a very high effect on the catalytic rate of these two deoxyribozymes. similar. The acceleration of Mn ²⁺ for the reaction is much greater than the other three ions. The acceleration of the other three ions for the catalytic reaction is M _g ²⁺ ~ Ca ²⁺ > Zn ²⁺ .

In the present invention, under single transformation conditions (50 mM Tris-HCl, pH 7.5, 50 mM Mg ²⁺ ), the catalytic rate constant was determined, some deoxyribozyme analogs were faster than the prototype 10-23 deoxyribozyme, and some deoxyribozyme analogs were slower (Table 6). The rate constant of the catalytic reaction of each deoxyribozyme analog is calculated according to the following formula. The final percent lysis of the reaction was set at 90%. The data used is the average of three independent real faces, and the error between these three results is less than 20%.

In the present invention, the base composition of the cleavage site of the substrate was changed, and a total of five substrate sequences (Table 7) were evaluated, and the catalytic rate changes of LKDZ21 and DZ01 were evaluated. They exhibit the same cutting behavior. The shear rates for AU and GU are close, while the shear rates for AC and GC are much lower. There is no shearing effect on the whole DNA substrate. Table 6 Catalytic Rate Constants for Deoxyribozyme Analogs Containing Nucleoside Analogs 1-4 and 6 Under Single Conversion Conditions

No. 10-23 Sequence composition of deoxyribozyme analogues

DZ01 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct)-3' 0.2362

LKDZ22 5'-d(tgc tct cca GGC TIG CTA CAA CGA cct gca cct)-3' 0.1161

LKDZ23 5'-d(tgc tct cca GGC TAG CT1 CAA CGA cct gca cct)-3' 0.5439

LKDZ24 5'-d(tgc tct cca GGC TAG CTA CIA CGA cct gca cct 3' 0.1186

LKDZ25 5'-d(tgc tct cca GGC TAG CTA CAA CGI cct gca cct)-3' 0.049

LKDZ26 5'-d(tgc tct cca GGC TAG CTA CA1 CGA cct gca cct)-3' 0.2044

LKDZ12 5'-d(tgc tct cca GGC T2G CTA CAA CGA cct gca cct>3' 0.015

LKDZ13 5'-d(tgc tct cca GGC TAG CTA CAA CG2 cct gca cct 3' 0.0171

LKDZ14 5'-d(tgc tct cca GGC TAG CT2 CAA CGA cct gca cct)-3' 1.4565

LKDZ15 5'-d(tgc tct cca GGC TAG CTA C2A CGA cct gca cct)-3' 0.0282

LKDZ16 5'-d(tgc tct cca GGC TAG CTA CA2 CGA cct gca cct)-3' 0.1676

LKDZ17 5'-d(tgc tct cca GGC TAG CTA CAA CG3 cct gca cct)-3' 0.1299 LKDZ18 5'-d(tgc tct cca GGC TAG CTA CA3 CGA cct gca cct)-3' 0.1551

LKDZ19 5'-d(tgc tct cca GGC TAG CTA C3A CGA cct gca cct)-3' 0.0923

LKDZ20 5'-d(tgc tct cca GGC T3G CTA CAA CGA cct gca cct)-3, 0.0193

LKDZ21 5'-d(tgc tct cca GGC TAG CT3 CAA CGA cct gca cct>3' 2.603

LKDZ27 5'-d(tgc tct cca GGC TAG CT4CAA CGA cct gca cct)-3' 0.7658

LKWQ-06 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct)-3' -

LKWQ-07 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct 3' 0.2454

LKWQ-08 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct>3' -

LKWQ-09 5'-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct)-3' 0.6363 Table 7 Sequence composition of five substrates

By comparing the effects of pH, metal ions, and different substrates on the catalytic rates of LKDZ21 and DZ01, we can preliminarily conclude that the catalytic mechanism is the same, 2,-OH acts as a nucleophile to attack the ortho-position of phosphorus atoms. 2,3,-cyclic phosphate and 5,-OH are the two products of the terminal. The increase in catalytic rate by the modified base of LKDZ21 may be due to the optimization of the conformation of the catalytic domain, thereby optimizing the spatial position of each group participating in the catalytic reaction and reducing the reaction energy barrier.

The respective deoxyribozyme analogs were cleaved with the unmodified 10-23 deoxyribozyme DZ01, and the results are shown in Figure 3-9.

Figure 3 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 1. Reaction conditions: Deoxyribozyme analog (2000 nmol) The substrate (20 nmol) was reacted in a buffer of Tris-HCl (50 mM, pH 7.5), 2 mM Mg ²⁺ . The sampling time points of DZ01 and LKDZ23 were 0 min, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h. The sampling time points of LKDZ22, LKDZ24, LKDZ25, LKDZ26 are: O min, 15 min, 30 min, 45 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h.

Figure 4 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 2. Reaction conditions: The deoxyribozyme analog (0.1 nmol) and the substrate (10 nmol) were reacted in a buffer of Tris-HCl (50 mM, pH 8.0), 50 mM Mg ²⁺ . Sampling time points for LKDZ12, LKDZ13, LKDZ15, LKDZ16: 0 min, 15 min, 30 min, 45 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, lOh. Sampling of LKDZ14 The time points are 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 75 min, 90 min, 105 min, 120 miii.

Figure 5 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 3. Reaction conditions: The deoxyribozyme analog (0.1 nmol) and the substrate (10 nmol) were reacted in a buffer of Tris-HCl (50 mM, pH 8.0), 50 mM Mg ²⁺ . Sampling time points for LKDZ17, LKDZ18, LKDZ19, LKDZ20: 0 min, 15 min, 30 min, 45 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h. The sampling time of LKDZ21 is 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 75 min, 90 min, 105 min, 120 min.

Figure 6 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 21. Reaction conditions: The deoxyribozyme analog (0.1 nmol) and the substrate (10 nmol) were reacted in a buffer of Tris-HCl (50 mM, pH 8.0), 50 mM Mg ²⁺ . Sampling time points: 0 h, 0.5 h, lh, 2 h, 3 h, 4 h, 6 h.

Figure 7 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 22. Reaction conditions: The deoxyribozyme analog (0.1 nmol) and the substrate (10 nmol) were reacted in a buffer of Tris-HCl (50 mM, pH 8.0), 50 mM Mg ²⁺ . Sampling time points: 0 h, 0.5 h, lh, 2 h, 3 h, 4 h, 6 h. Figure 8 shows a comparison of the cleavage reaction of 10-23 deoxyribozyme DZ01 and a deoxyribozyme analog containing deoxyadenosine analog 23. Reaction conditions: Deoxyribozyme analogue (0.1 nmol) and substrate (10 nmol) in Tris-HCl (50 mM, pH 8.0), 50 mM Mg ²⁺ buffer. Sampling time: 0 h, 0.5 h, lh, 2 h, 3 h, 4 h, 6 h.

Figure 9 shows a comparison of the cleavage activity of the deoxyribozyme analogs LKWQ06 (b)-LKWQ09 (e) and DZ01 (a) containing deoxyguanosine analogue 11 under single conversion conditions. Reaction reaction: The ratio of deoxyribozyme analog (2000 nmol) to substrate (20 nmol) was 100:1, and it was reacted in a buffer of Tris-HCl (50 mM, pH 7.5), 2 mM Mg ²⁺ . Sampling time points: O h, 0.5 h, lh, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 ho

The present invention modifies the catalytic domain of 10-23 deoxyribozyme to obtain a more efficient deoxyribozyme analog. This type of deoxyribozyme analog also retains the properties of the prototype 10-23 deoxyribozyme, which utilizes its recognition arm for highly specific recognition of the substrate. The recognition domains at both ends of the prototype 10-23 deoxyribozyme are designed based on the target gene fragment, and the catalytic domain remains unchanged. It has been used for the study of various pathogenic genes such as HIV, HBV, HCV and other viruses. Multiple gene segments, and multiple tumor gene segments. Therefore, the deoxyribozyme analog of the present invention can also be designed for any target gene fragment, and as long as the sequence composition of the target RNA is known, the corresponding deoxyribozyme analog can be designed and efficiently cleaved. Therefore, such deoxyribozyme analogs, like the prototype deoxyribozyme, can be used for genetic manipulation of any gene of interest, or as a candidate for gene therapy for disease-causing genes. In addition to the fragment of the vascular endothelial growth factor receptor mRNA in the embodiment, it may be an mRNA fragment selected from other tumor genes, a viral gene, or a full-length mRNA, or a target mRNA in a cell, or an animal. mRNA. Therefore, it will be a new class of drug candidates for broad-spectrum gene therapy.

The novel 10-23 deoxyribozyme analogs of the present invention are based on the structure of the prototype 10-23 deoxyribozyme to obtain certain advantages over the prototype 10-23 deoxyribozyme, the art of the art. The person skilled in the art can see through the technology of the present invention The 10-23 deoxyribozyme analog with the desired advantages is fully available. It is appreciated that the role of the prototype 10-23 deoxyribozyme against pathogenic genes, as well as related research, and the like, are useful for understanding the advantages and uses of the novel 10-23 deoxyribozyme analogs of the present invention. References to the role of the prototype 10-23 deoxyribozyme against pathogenic genes and related studies are listed below, the contents of which are incorporated herein by reference:

1. Nunamaker E. Α·, Zhang H.-Y., Shirasawa Y., Benoit, JN, Dean, DA Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression Am. J. Physiol Heart Circ, Physiol 2003, 285, H2240-H2247.

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DNazyme-mediated cleavage of Twist transcripts and increase in cellular apoptosis Biochem. Biophys. Res. Commun. 2003, 300, 17»-181.

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Iversen P. 0., Emanuel P. D., Sioud M. Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth Blood, 2002, 99, 4147-4153.

Schubert S. and Kurreck J. Ribozyme- and Deoxyribozyme-Strategies for Medical Applications Current Drug Γα ·^, 2004, 5, 667-681

Toyoda T., Imamura Y., Takaku H., Kashiwagi T., Hara-K., Iwahashi J., Ohtsu Υ·, Tsumura N., Kato H., Hamada N. Inhibition of influenza virus replication in cultured cells by RNA -cleaving DNA enzyme FEBS Lett 2000, 481, 113-116.

Mitchell A., Dass C. R" Sun L.-Q., Khachigian, LM Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1 Nucleic Acids Res. 2004, 32, 3065-3069. Kuwabara Τ·, Warashina M., Tanabe, T., Tani K., Asano, S., Taira K. Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA Nucleic Acids Res. 1997, 25, 3074-3081

Fahmy R. G" Waldman A., Zhang G., Mitchell A" Tedla N" Cai H, Geczy CR, Chesterman CN, Perry M., Khachigian LM Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun Nat Biotech. 2006, 24, 856-863.

Trepanier JB, Tanner JE, Alfieri C. Reduction in intracellular HCV RNA and virus protein expression in human hepatoma cells following treatment with 2 '-Om ethyl-modified anti-core deoxyriboz me Virology 2008, 377 339-344.

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[13] Brownl T. S., Chadalavada D. M., Bevilacqual P. C. Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength . MoL Biol. 2004, 341, 695-712.

[14] Sheng J" Al-Anouti F., Ananvoranicb S. Engineered delta ribozymes can simultaneous knock down the expression of the genes encoding uracil phosphoribosyltransferase and hypoxanthine-xanthine-guanine phosphoribosyltransferase in Toxoplasma gondii Inter. J. Parasitology 2004, 34, 253 -263.

[15] Coppins RL, Silverman S. K" Rational modification of a selection strategy leads to deoxyribozymes that Create native 3'-5: RNA linkages J. Anu Chem, Soc. 2004, 126, 16426-16432.

[16] Santoro S. W., Joyce G F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl Acad. Scu USA 1997, 94, 4262-4266.

[17] Sidorov A. V., Grasby J. A., Williams D. M. Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities Nucleic Acids Res. 2004, 32, 1591-1601.

[18] Ting R., Thomas JM, Lermer L., Perrin DM Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation Nucleic Acids Res. 2004, 32, 6660-6672.

[19] May JP, Ting R" Lermer L., Thomas JM, Roupioz Υ·, Perrin DM Covalent schiff base catalysis and turnover by a DNAzyme: a M ²⁺ -independent AP-endonuclease mimic J, Am. Chem, Soc, 2004, 126, 4145-4156.

[20] Gourlain T., Sidorov A., Mignet N., Thorpe S., Lee SE, Grasby JA, Williams DM Enhancing the catalytic repertoire of nucleic acides. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR Nucleic Acids Res. 2001, 29, 1898-1905.

[21] Santoro S. W., Joyce G. F., Sakthivel K, Gramatikova S., Barbas, III C. F. RNA cleavage by a DNA enzyme with extended chemical functionality J. Am. Chem. Soc, 2000, 122, 2433-2439.

[22] Bashkin JK, Frolova EI, Sampath U. Sequence-specific cleavage of HIV mRNA by a ribozyme mimic J. Anu Chem. Soc. 1994, 116, 5981-5982. [23] Kubo Τ·, Takamori Κ·, Kanno Κ·, Rumiana Β·, Ohba Η·, Matsukisono Μ·, Akebiyama Υ·, Fujii M. Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes Bioorg. & Med. Chenu Lett 2005, 15, 167-170.

Claims

Rights request

1. 10 " 2 1- A4C A? 2 10-23 deoxyribozyme analog, which is represented by the following formula:

3'- N! N ₂ N ₃ N ₄ N ₅ N ₆ ... Ni X _1S X"C ₁₃ X ₁₂ X„C ₁₀ X ₉ X ₈ C ₇ X^X

C3X2X1 RNi+i7 Ni+is i+i9 i+2o N _n -5 »

Or as shown below:

3'- Ν! Ν ₂ Ν ₃ Ν ₄ Ν ₅ N _i+18 Ν i+19 Nj+20 Ν _η -5,

₉ x ₈ c ₇

among them,

Representing the recognition moiety at both ends of the 10-23 deoxyribozyme analog, and the number of base groups at both ends is the same or different, each independently being 4 to 25;

3'- X15X14C13 Xi ₂ XnCio X9X8C7 X6X5X4 C3X2X1 -5, is the catalytic domain;

n is an integer from 4 to 50;

i is an integer of 4-33;

15X i

14X x ₂

13C c ₃

₁₂ xx ₄

11X x ₅

10C x ₆

The catalytic domain portion ₃ xx ₈ c ₇ is a 10-23 deoxyribozyme catalytic structure c ₃

τ ₄

Α ₅

Domain part ₃ i, 2, 4, 5, 6, 8, 9, 11, 12. 14, 15 in AT ₈ C ₇ Any one or more of the residue residues are each independently selected from the following formula: 1, formula 0,

In the nucleoside analogs of the above formula J, formula 8, formula 0, formula E and formula F, each substituent is independently defined as follows:

When z is a subunit, R ^{1 in} position 7 is independently selected from the group consisting of: hydrogen, halogen (eg, fluorine, chlorine, bromine, iodine), pseudohalogen (eg, sulfur ^ J , , amide group (eg, CONH ₂ , CONHR ⁷⁾ , CONR ⁷ ₂ ), C ₆ _ ₂ .Aromatic, heteroaromatic and heterocyclic structures, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, a C ₆ _.20 aromatic group, a C ₃ .io heteroaryl group and a heterocyclic structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), anthracenyl, substituted anthracene Base HC(NR ⁷ ₂ )=NR ⁷ , 31⁄2, SR ⁷ , CONH ₂ , CO HR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg cyano, thiocyano) Carboxyl group,

L is a tether selected from the group consisting of Cw. Linear or branched alkyl arms (eg, d- ₆ straight or branched alkyl arms, such as fluorenylene, 1,2-ethylene, trisino, tetramethylene), C _M0 unsaturated alkyl An arm (for example, a C ₂ _ ₄ unsaturated alkyl group such as an olefin bond or an acetylenic bond) or a C ₃ . _{1 ()} cycloalkyl arm, the linking arm may also be an amide bond, an ester bond, an ether bond, or a thiol bond. Straight and branched structure,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a C straight or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl) Base, hexyl), C ₂ . _{1 ()} unsaturated fluorenyl (eg, C ₂ _ ₄ unsaturated alkyl, such as ethenyl, propenyl, ethynyl, propynyl). Cycloalkyl (e.g. C _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring,

(2) The 2-position substituents R ^{2 of the} purine nucleoside analogs J, B, D, E are each independently selected from the group consisting of: hydrogen, hydroxy, thiol, halogen (eg, fluorine, chlorine, bromine, reef), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , HCOR ⁷ (aminoacyl), fluorenyl, SR ⁷ , C ₆ _ ₂ . An aryl group, a C ₃ _-8 heteroaryl group and a heterocyclic ring structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, a C ₆ _.20 aromatic group, a C ₃ .io heteroaryl group and a heterocyclic structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), anthracenyl, substituted anthracene Base NH-C(NR ⁷ ₂ )=NR ⁷ , fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen (eg cyano, thiocyano) ), carboxyl group,

L is a connecting arm selected from the following: a linear or branched guanidine arm (for example, a C straight or branched alkyl arm such as an anthranylene group, a 1,2-ethylene group, a trimethylene group, a tetramethylene group) or a C ₂ _.10 unsaturated alkyl group a base arm (such as an olefinic bond, an acetylenic bond), a C ₃ _.10 cycloalkyl arm (for example, a C ₃ _-6 cycloalkyl group), and the linking arm may also be an amide bond, an ester bond, an ether bond, or a thioether bond. Chain and branch structure,

R ^{7 is} each independently selected from: a straight or branched alkyl group (eg, a C" straight or branched alkyl group, such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert. Butyl, pentyl, hexyl), C ₂ unsaturated alkyl (eg unsaturated alkyl such as vinyl, propenyl, ethyl, propynyl), C ₃ _{.1 ()} cycloalkyl (eg C _{_3.6} cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring, the aromatic group and the heteroaromatic group optionally substituted with one or more R ^{9 is} substituted, and R ^{9 is} the same as R ⁷ ;

(3) The substituents R ^{3 of the} purine nucleoside analogs J and B are each independently selected from the group consisting of: hydrogen, ^&, hydroxy, halogen, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), Sulfhydryl, sulfhydryl, SR ⁷ , C ₆ _ ₂ . Aromatic group, C ₃ . ₂ . Heteroaryl and heterocyclic structures, R ⁷ , or LR ⁸ , where:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, a C ₆ _.20 aromatic group, a C ₃ ., an o heteroaryl group and a heterocyclic ring structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), anthracenyl, substituted Sulfhydryl NH-C(NR ⁷ ₂ )=NR ⁷ , sulfhydryl, SR ⁷ , CO H ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg, aryl, sulphur) Cyano), carboxyl,

L is a tether selected from the group consisting of: Cwo straight or branched alkyl arms (eg, C straight or branched alkyl arms, such as anthracenylene, 1,2-ethylene, triamylene, tetramethylene group), C _2. ₁₀ arm unsaturated alkyl (e.g. cockroach bond, acetylene bond), C ₃ .io arm cycloalkyl (e.g. C _3. ₆ cycloalkyl), containing the connecting arm may also be an amide bond, an ester a linear and branched structure of a bond, an ether bond, or a thioether bond,

R ^{7 is} each independently selected from. a linear or branched alkyl group (for example, a d- ₆ straight or branched alkyl group such as decyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl) , c ₂ . _{1 ()} unsaturated alkyl (eg, c ₂ ₄ unsaturated alkyl, such as vinyl, propyl, ethynyl, propynyl), c ₃ - _ie cycloalkyl (eg, c ₃ . _{a 6-} cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group, and a linear and branched structure containing an aromatic ring,

(4) The purine nucleoside analogs J and D at the 8-position W may each independently be a carbon atom or a nitrogen atom, wherein:

When w is ^^ neutrons, which being optionally R ¹ "substituents, which takes ^ J ^ R ^1" are each independently selected from: hydrogen, halogen (e.g. fluorine, chlorine, bromine, iodine), pseudohalide (e.g. H^, sulfur

^^, COOR ⁷ (ester group), CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ (amido group), J. fluorenyl group, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl group), sulfhydryl group, SR ⁷ , C ₆ _ ₂ . Aromatic base. a heteroaromatic and heterocyclic structure, R ⁷ , LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and a _ _{2 ()} aryl group. Heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ > NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen

(eg fluorine, chlorine, bromine, hydrazine), pseudohalogen (eg n-group, thiocyano), carboxyl group,

L is a connecting arm selected from the following: a linear or branched alkyl arm (for example, a linear or branched alkyl arm such as methylene, 1,2-ethylene, trisino, tetradecyl), C ₂ _ ₁₀ An unsaturated sulfhydryl arm (for example, a C ₂ _4 unsaturated sulfhydryl arm, such as a gynecological bond, an acetylenic bond), a C ₃ . _{1 ()} cycloalkyl arm (for example, a c ₃ _ ₆ ring 垸 base arm), which may also be a linear and branched structure containing an amide bond, an ester bond, an ether bond, or a thioether bond,

R ^{7 is} each independently selected from. A linear or branched alkyl group (for example, a linear or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl). Unsaturated; ^ (for example C ₂ _ ₄ unsaturated alkyl group, such as ethyl hydrazino, propenyl, ethynyl, propynyl). a cycloalkyl group (for example, a c ₃ _-6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group), and a linear and branched structure containing an aromatic ring,

When W is a nitrogen atom, it is not substituted;

(5) Adenosine analogues B and E, Z and V are in a five-membered ring structure, and Z and V are each independently carbon, nitrogen, oxygen, sulfur, etc.

When z is ^^, the R ¹ thereon is independently selected from the group consisting of: hydrogen, halogen (eg, fluorine, chlorine, bromine, iodine), pseudohalogen (eg, *J>, sulfur*J, , amide group ( For example, CONH ₂ , CONHR ⁷ , CO R ⁷ ₂ ), € ₆ _ ₂₀ aryl, C ₃ _ _2. heteroaryl or heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of hydroxyl, amino, Q.20 aryl, C ₃ _.10 heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , fluorenyl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg, sulfur ^ J^), ,

L is a connecting arm selected from the following: a linear or branched alkyl arm (for example, a C straight or branched alkyl arm such as methylene, 1,2-ethylene, trisino, tetradecyl), C ₂ _.10 unsaturated alkyl a base arm (eg, a C ₂ -4 unsaturated alkyl arm, such as an olefinic bond, an acetylenic bond). a cycloalkyl arm (for example, a C ₃ _-6 cycloalkyl arm), which may also be a linear or branched structure containing an amide bond, a S bond, an ether bond, or a thioether bond.

R ^{7 is} each independently selected from ( ^ straight or branched alkyl (for example, C straight or branched alkyl) Bases such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl). An unsaturated alkyl group (for example, a C 4 unsaturated alkyl group such as a vinyl group, a propenyl group, an ethynyl group, a propyl group). Cycloalkyl group (e.g., c _3. ₆ cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), and linear and branched structures containing an aromatic ring,

When Z is a nitrogen atom, it is taken as R ¹ but does not include halogen and pseudohalogen.

When V is ^^, each of them is optionally replaced by ^J^ R ¹¹ ,

R ^{11 has} the same definition as R ¹ ; when V is a nitrogen atom, R ^{11 has} the same definition as R ¹ but is not substituted by halogen or pseudohalogen;

When Z and V are oxygen or a sparing atom, there is no substituent;

(6) a pyrimidine nucleoside analog F, wherein the substituents R ⁴ and R ^{5 at the 5-} and 6-positions are each independently selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, iodine), pseudohalogen (alkyl group, thiocyanate) Base, carboxyl group, COOR ⁷ (ester group), CO H ₂ , CONHR ⁷ , CONR ⁷ ₂ (amide group), NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), sulfhydryl, SR ⁷ , C ₆ _ ₂ . Aromatic and. a heteroaromatic and heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of hydroxyl, amino, Q.20 aryl, C ₃ , o heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , NHCOR ⁷ (aminoacyl), fluorenyl, substituted anthracene Base NH-C (R = NR ⁷ , sulfhydryl, SR ⁷ , CONH ₂ , CONHR ⁷ , CONR ⁷ ₂ , halogen (eg fluorine, chlorine, bromine, reef), pseudohalogen (eg cyano, thiocyano), carboxyl ,

R ^{7 is} each independently selected from. A linear or branched alkyl group (for example, a linear or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl). An unsaturated alkyl group (e.g., a C ₂ - ₄ unsaturated alkyl group such as a vinyl group, a propenyl group, an ethynyl group, a propynyl group). a cycloalkyl group (for example, a C ₃ _-6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group), and a linear and branched structure containing an aromatic ring,

L is a Cwn straight or branched alkyl arm selected from the group consisting of a d. ₆ linear or branched alkyl arm, such as methylene, 1,2-ethylene, triammonium, tetraammine Base), C _M0 An unsaturated alkyl arm (eg, an unsaturated alkyl arm, such as a female bond, a bulk bond), a C ₃ . _{1 ()} cycloalkyl arm (eg, a C ₃ ₆ cycloalkyl arm), which may also be an amide containing bond , ester bonds, ether bonds, linear and branched structures of thioether bonds,

(7) Nucleoside analogs J, B, D, E, F, the sugar ring moieties thereof are each independently selected from the group consisting of deoxyribosyl, other five-membered sugar ring groups, six-membered sugar ring groups, LNA type, or other modifications. Sugar ring structure [Preferably, the sugar ring portions are each independently selected from the group consisting of a deoxyribosyl group, a six-membered sugar ring group, and an LNA type], and the configurations of the sugar ring portions are each independently a D- or L-form, wherein

When the sugar ring moiety is a five member sugar ring, the 2,-position substituents R ⁶ are each independently selected from the group consisting of hydrogen, amino, fluorine atom, methoxy group, ethoxy group, propyl L group, 曱H-vinyloxy group. , Ethyl vinyl H group, propyl vinyl L-group, decylamino group, dimethylamino group, ethylamino group, diethylamino group, propylamino group, dipropylamino group, cyclopropylamino group.

2. The 10-23 deoxyribozyme analog of claim 1, wherein N represents the recognition moiety at both ends of the 10-23 deoxyribozyme analog, and the number of both ends is the same or different, each independently from 4 to 25, and The 耙 sequence targeted is paired with Watson-Crick.

The 10-23 deoxyribozyme analog according to claim 1 or 2, which is characterized by any one or more of the following:

a) n is an integer from 4 to 50, or n is an integer from 10 to 40, or n is an integer from 15 to 40, or n is an integer from 20 to 40, or n is an integer from 4 to 40;

b) i is an integer of 4-33, or i is an integer of 4-15, or i is an integer of 4-12, or i is an integer of 6-12;

c) the catalytic domain portion is in the catalytic domain of the 10-23 deoxyribozyme Any one of the residues 1, 2, 4, 5, 6, 8, 9, 11, 12, 14, or 15 is selected from the cores of Formula J, Formula 8, Formula 0, Formula E, and Formula F Substituted for a glycoside analog, or the catalytic domain portion is the first, 2, 4, 5, 6, 8, 9, 11, 12, 14, or 15 of the 10-23 deoxyribozyme catalytic domain portion Any two or more of the residues are substituted with a nucleoside analog selected from Formula J, Formula 8, Formula, Formula E, and Formula F;

d) the catalytic domain portion is any one of residues 5, 9, 11, 12, or 15 in the 10-23 deoxyribozyme catalytic domain portion selected from the core of Formula J or Formula B The glycoside analog is substituted, or the catalytic domain portion is any one of residues 1, 2, 6, or 14 in the 10-23 deoxyribozyme catalytic domain portion selected from Formula D or Formula E. a nucleoside analog substitution; or the catalytic domain portion of the 10-23 deoxyribozyme catalytic domain portion of any of residues 4, or 8 is substituted with a nucleoside analog selected from formula F .

The 10-23 deoxyribozyme analog according to any one of claims 1 to 3, which is characterized by any one or more of the following:

a) the catalytic domain portion is a nucleoside analog selected from the group consisting of residues 5, 9, 11, 12, or 15 in the 10-23 deoxyribozyme catalytic domain portion selected from the group consisting of ;

b) the catalytic domain portion is any one of residues 1, 2, 6, or 14 in the 10-23 deoxyribozyme catalytic domain portion substituted with a nucleoside analog selected from the group consisting of:

The catalytic domain portion of mc) is the first analog substitution in the 10-23 deoxyribozyme catalytic domain portion:

The 10-23 deoxyribozyme analog according to any one of claims 1 to 4, which is characterized by any one or more of the following:

a) wherein, in the nucleoside analog of formula 0, formula E, Z is each independently selected from carbon and nitrogen atoms, wherein

When Z is ^^ midnight, taking 7 ^ J ^ R ¹ is independently selected from: hydrogen, halogen (e.g. fluorine, chlorine, bromine, iodine), C ₆ _{20 is} an aromatic group (e.g., C ₆ aromatic group, e.g. Phenyl), C ₃ .10 heteroaryl and heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of hydroxyl, C ₆ _ ₂ . An aromatic group (for example, a C ₆ aromatic group such as a phenyl group), a C ₃ .io heteroaryl group and a heterocyclic structure (for example, a C ₃ _-8 heteroaromatic group such as a pyridyl group, a 2- or 4-imidazolyl group), OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, ),

L is a connecting arm selected from the following: a linear or branched alkyl arm (e.g., a linear or branched alkyl arm such as methylene, 1,2-ethylene, trisino, tetramethylene),

R ^{7 is} each independently selected from C _M . a linear or branched alkyl group (for example, a C straight or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl), a linear and branched structure containing an aromatic ring;

b) in the nucleoside analog of the formula 8, formula 0, formula E, the 2-position substitution The radicals R ² are each independently selected from the group consisting of: hydrogen, amino, hydroxy, thiol, C ₆ _ ₂ . Aromatic, C ₃ .io heteroaryl and heterocyclic structures, halogen (eg fluorine, chlorine, bromine, ), OR ⁷ , NHR ⁷ , R ⁷ ₂ , NHCOR ⁷ (aminoacyl), SR ⁷ , R ⁷ , or LR ⁸ , where:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ _ ₂ . Aromatic group, C ₃ _ ₁₍₎ heteroaryl and heterocyclic structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , SR ⁷ , fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg, aryl, thiocyano), carboxyl,

L is a tether selected from the group consisting of C _w . a linear or branched 垸-arm (eg, a C" linear or branched 垸-based arm, such as methylene, 1,2-ethylene, tri-indenyl, tetramethylene),

R ^{7 is} each independently selected from: Cwn straight or branched alkyl (eg, d- ₆ straight or branched alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, a tert-butyl group, a pentyl group, a hexyl group, a linear and branched structure containing an aromatic ring;

c) the nucleoside analogues of formula B in the formula, the substituents R ³ 6 are each independently selected from: hydrogen, ■ ½, hydroxy, guanidino, ^{^{halo, OR 7, NHR 7, NR}} 7 2, SR ⁷ , C ₆ _ ₂ . Aromatic base. Heteroaryl and heterocyclic structures, R ⁷ , or LR ⁸ . among them:

R ⁸ is selected from hydroxy, amino, <: _₆₂ "aromatic group, _{_<31 ()} and heterocyclic aromatic hetero ^{^{structure, OR 7, NHR 7, R}} 7 2, guanidino, substituted guanidino HC ( NR ⁷ ₂ )=NR ⁷ , SR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine),

L is a connecting arm selected from the following: a linear or branched alkyl arm (eg, a d- ₆ straight or branched alkyl arm such as methylene, 1,2-ethylene, trisino, tetramethylene),

R ^{7 is} each independently selected from Cwo straight or branched alkyl (eg, d- ₆ straight or branched alkyl such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert. Butyl, pentyl, hexyl), linear and branched structures containing aromatic rings;

d) In the nucleoside analog of the formula J and formula D, the 8 positions W may each independently be a carbon atom or a nitrogen atom, wherein:

When w is a sub-substituent, it is optionally substituted with R ¹ ", and each of R & R ^{1 () is} independently selected from the group consisting of: hydrogen, halogen (e.g., fluorine, chlorine, bromine, hydrazine), heteroaromatic and hetero Ring structure, amino group, sulfhydryl group, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , R ⁷ , or LR ⁸ , wherein: R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, a C ₆ — ₂₀ aromatic group, a C ₃ — _ie heteroaryl group, and a heterocyclic structure, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , an indenyl group, a substituted indenyl group HC (NR ⁷ ₂ ) =NR ⁷ , SR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine),

L is a tether selected from the group consisting of: C _M o a straight or branched guanidine arm (for example, a linear or branched alkyl arm such as methylene, 1,2-ethylene, triamyl, tetra methyl),

R ^{7 is} each independently selected from CM. a linear or branched alkyl group (for example, a C straight or branched alkyl group such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl), a linear and branched structure containing an aromatic ring;

e) wherein the nucleoside analogs of formula B and formula E are each independently a carbon atom, which is taken as R ¹ , and R ¹ are each independently selected from the group consisting of: hydrogen, halogen (eg, fluorine, chlorine, bromine, reef),

. a heteroaromatic and heterocyclic structure, a carboxyl group, NHR ⁷ , NR ⁷ ₂ , R ⁷ , LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of hydroxyl, amino, C ₆ . ₂₀ aryl, C ₃ _.10 heteroaryl and heterocyclic structures, OR ⁷ , NHR ⁷ , NR ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C (NR ⁷ ₂ ) = NR ⁷ , SR ⁷ , halogen (eg fluorine, chlorine, bromine, angstrom),

L is a connecting arm selected from the following: a linear or branched alkyl arm (for example, a d. ₆ linear or branched alkyl arm such as an anthranylene group, a 1,2-ethylene group, a trimethylene group, a tetramethylene group),

R ^{7 is} each independently selected from C" ₀ straight or branched alkyl groups (eg, C straight or branched alkyl such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, a tert-butyl group, a pentyl group, a hexyl group, a linear and branched structure containing an aromatic ring;

When Z is a nitrogen atom, the substituent is R ¹ , but does not include a halogen and a pseudohalogen substituent;

f) In the nucleoside analog of the formula E, each of V is independently a carbon atom or a nitrogen atom. When ¥ is midnight, which is optionally substituted by taking ¹¹ R, R ¹¹ are each independently selected from: hydrogen, halogen (e.g. fluorine, chlorine, bromine, reef), C ₆ _ _2. An aryl group, a C ₃ _.10 heteroaryl group and a heterocyclic structure, R ⁷ , or LR ⁸ , wherein:

R ^{8 is} selected from the group consisting of a hydroxyl group, an amino group, and C ₆ _ ₂ . Aromatic base. Heteroaryl and heterocyclic structures, OR ⁷ , NHR NR ⁷ 2, fluorenyl, substituted fluorenyl NH-C(NR ⁷ ₂ )=NR ⁷ , SR ⁷ , halogen (eg fluorine, chlorine, bromine, iodine), pseudohalogen (eg cyano, thiocyano),

L is a tether selected from the group consisting of Cw. a linear or branched alkyl arm (for example, a d. ₆ linear or branched alkyl arm such as an anthranylene group, a 1,2-ethylene group, a triadenylene group, a tetramethylene group),

R ^{7 is} each independently selected from dw straight or branched (eg, C straight or branched alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, a pentyl group, a hexyl group, a linear and branched structure containing an aromatic ring,

When V is a nitrogen atom, it is optionally substituted with ^J^ R ¹¹ , but does not include halogen and pseudohalogen.

g) In the nucleoside analog of formula F, the substituents R ⁴ and R ^{5 at} positions 5 and 6 are each independently selected from the group consisting of hydrogen, halogen (fluorine, chlorine, bromine, reef), C ₆ . ₂ . An aromatic group (for example, a C ₆ aromatic group such as a phenyl group), a C ₃ _ _{1 ()} heteroaryl group and a heterocyclic structure (for example, a C ₃ _-8 heteroaryl group, for example, a pyridyl group, an imidazolyl group), R ⁷ , or LR ⁸ , where:

R ⁸ is selected from hydroxy, amino, C _6. ₂₀ aryl (e.g., C ₆ aryl group, e.g. phenyl), C _3. ₁₀ heteroaromatic group, and a heterocyclic ring structure (e.g., C _3. ₈ heteroaryl group, e.g., pyridine Base, imidazolyl), OR ⁷ , NHR ⁷ , R ⁷ ₂ , fluorenyl, substituted fluorenyl NH-C(R ⁷ ₂ )=NR ⁷ , SR ⁷ , halogen (eg fluorine, chlorine, bromine, hydrazine),

R ^{7 is} each independently selected from Cwo straight or branched alkyl (eg, C straight or branched alkyl such as fluorenyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl) Base, pentyl, hexyl), linear and branched structures containing aromatic rings,

L is a connecting arm selected from the following: a linear or branched thiol arm (eg, a C straight or branched alkyl arm, such as an anthranylene, 1,2-ethylene, trimethylene, tetradecyl), h) In the nucleoside analogs of Formula 0, Formula E and Formula F, the sugar ring moieties thereof are each independently selected from the group consisting of deoxyribosyl groups, other five-membered sugar ring groups, LNA type, and six-membered sugar ring groups [preferably, The sugar ring moieties are each independently selected from the group consisting of deoxyribosyl groups and LNA types.

6. The 10-23 deoxyribozyme analog according to any one of claims 1 to 5, which can be used The nucleoside analogs of the involved nucleoside analogs J, B, D, E, F modify the five dA sites X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ :

(1) In the modification of 5 dA sites X ₅ , X ₉ , X _n , X ₁₂ , X ₁₅ , compounds J, B, D, E, F can be inserted into these sites instead of dA;

(2) In the modification of 5 dA sites X _s , Χ ₉ , Χ π, Χ ₁₂ , Χ ₁₅ , it is preferred to select compound J and hydrazine instead of dA;

(3) In the modification of the five dA sites X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ , they are all positions which are subcutaneously replaceable;

(4) In the modification of the five dA sites X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ , in the single substitution modification, X ₉ is a preferred modification site;

(5) In the modification of 5 dA sites Χ ₅ , Χ ₉ , Χ _π , Χ ₁₂ , Χ ₁₅ , multiple sites ( 2-5 ) can be modified at the same time.

7. The 10-23 deoxyribozyme analog according to any one of claims 1 to 6, wherein the X ₂ , X , X " at the 4 dG positions are each independently optionally selected by the nucleoside analog J , B, D, E, F substituted positions, where:

(1) In the position of 4 dG, in the modification of X" X ₂ , Xe, X ₁₄ , compounds J, B, D, E, F can be inserted into these sites instead of dG;

(2) in the position of 4 dG, Χ ₁₅ Χ ₂ , Χβ, Χ ₁₄ modification, preferably select compound D and Ε insert these sites instead of dG;

(3) In the modification of 4 dG positions, Χ ₂ , Χβ, Χ ₁₄ , in the case of single substitution modification, G2 and G14 are preferred modification sites;

(4) In the modification of 4 dG positions, Χ ₂ , Χί, Χ ₁₄ can modify multiple sites at the same time.

8. The 10-23 deoxyribozyme analog according to any one of claims 1 to 7, wherein the positions x4 and Xs of the two dTs are each independently optionally optionally referred to by the nucleoside analogs J, B, The position substituted by D, E, F, where:

(3) In the modification of the position of two dTs and X ₈ , it is preferred to select the compound F to insert these sites instead of dT.

9. The 10-23 deoxyribozyme analog according to any one of claims 1 to 8, which utilizes the above nucleoside analogs J, B, D, E, F to modify the domain of the deoxyribozyme, 5 dA Modification sites (X ₅ , X ₉ , X„, X ₁₂ , X ₁₅ ), 4 dG modification sites (XX ₂ , X6, X ₁₄ ), and two dT modification sites (X4 and X) ₈ ), which can be substituted by a combination of a plurality of different types of nucleoside analogs.

10. The 10-23 deoxyribozyme analog according to any one of claims 1 to 9, wherein the single or combined substitution of the nucleoside analog of formula J, formula 0, formula 式, formula F is also related to the natural base Deletions (eg, deletion of T8) - modify the catalytic domain of 10-23 deoxyribozyme.

11.1 1010-23 deoxyribozyme to any one of claims analogue, two positions which X ₉ and X ₁₄ are each independently optionally nucleoside analogs are involved J, B, D, E, F is replaced.

12. The 10-23 deoxyribozyme analog according to any one of claims 1 to 11, wherein the single or combined substitution of the nucleoside analogs J, B, D, E, F can be used to increase the stability of the deoxyribozyme analog. The combination of modifications. The method for protecting against nucleic acid oxime is a thiolatate bond skeleton; 2,-fluoro, V- 曱H, 2,-曱乙埽Λ(ΜΟΕ), 2,-Blli 2,-acetamidine , LNA, etc.; introduction of inverted nucleomonomers at E3,-end to obtain enzyme stability A newer novel deoxyribozyme analog. The modification described above for improving the stability of the deoxyribozyme can be used for both the catalytic domain of the deoxyribozyme and the recognition domain of both ends thereof.

13. The 10-23 deoxyribozyme analog according to any one of claims 1 to 12, wherein the introduction of the nucleoside analog of formula 8, formula D, formula E, formula F can improve the transport of the deoxyribozyme analog The combination of modifications performed. Among the methods for improving transport include liposomes and cationic liposomes, and encapsulation of other transport materials; covalent attachment of cholesterol, PEG, etc. to deoxyribozymes.

The 10-23 deoxyribozyme analog according to any one of claims 1 to 13, which is a 10-23 deoxyribozyme analog selected from the group consisting of the following:

LKDZ03 5'-d(tgc tct cca GGC TAG C22A CAA CGA cct gca T8=22 cct)-3'

LKDZ04 5'-d(tgc tct cca GGC 23AG CTA CAA CGA cct gca T4=23

Cct)-3,

LKDZ05 5'-d(tgc tct cca GGC TAG C23A CAA CGA cct gca T8=23

Cct)-3,

Wherein, "the position of the modified monomer indicates the type of the monomer to be substituted and the position of the monomer, and the "nucleoside analog monomer number" indicates a nucleoside analog monomer selected from the following numbers:

1 : R ¹ = H 6: R ¹ = H 11 : R ¹ = H

2: R ¹ = (CH ₂ ) ₃ OH 7: R ¹ = (CH ₂ ) ₃ OH 12: R ¹ = (CH ₂ ) ₃ OH

3: R ¹ = (CH ₂ ) ₃ NH ₂ 8: R ¹ = (CH ₂ ) ₃ NH ₂ 13: R ¹ = (CH ₂ ) ₃ NH ₂

4: R ¹ — CH CH CgHg 9: R ¹ = CH2CH2CQH5 14: R ¹ = CH 2C H2C5H

5: R ¹ = CH ₂ CH ₂ (4-) lm 10: R ¹ = CH ₂ CH ₂ (4-) lm 15: R ¹ = CH ₂ CH ₂ (4-) ln

16: R ¹ = H 21: R ⁴ = CH ₂ OH

17: R ¹ = (CH ₂ ) ₃ OH 22: R ⁴ = CH ₂ CH ₂ OH

18: R ¹ = (CH ₂ ) ₃ NH ₂ 23: R ⁴ = CH ₂ CH ₂ CH ₂ OH

19: R ¹ = CH ₂ CH ₂ C ₆ H ₅ 24: R ⁴ = CH ₂ CH ₂ (4-) lm

20: R ¹ = CH ₂ CH ₂ (4-)lm

A method for producing a 10-23 deoxyribozyme analog according to any one of claims 1 to 14, which is a solid phase synthesis of a 10-23 deoxyribozyme analog by a phosphoramidite method.

16. The method of preparation of claim 15 directed to the sequence of the following substrates:

5'- N N'2 N'3 N' ₄ N' ₅ N' ₆ ..···· N'| RY N' _i+3 N' _i+4 N' _i+5 N' _i+6 . .·· ·· N' _m -3', Synthesis of the corresponding 10-23 deoxyribozyme analog:

3'- Ni N2 N3 N4 N ₅ N ₆ ·.. ,.· Nj R Nj+17 Ni+is Nj ₊ ig Nj+20 .·· ··· N _n -5'

θ χ ₈ 〇7 where,

N, which is the nucleotide monomer composition of the substrate, and N is the nucleotide monomer composition of the recognition domain of 10-23 deoxyribozyme. R is a purine nucleotide unit, Y is a pyrimidine nucleotide unit, i is an integer of 4-33, and m is the number of monomers of a partial sequence or a full-length sequence for genetic manipulation and gene therapy (for example, 6-1000) An integer, such as an integer from 6 to 800, such as an integer from 6 to 500, such as an integer from 6 to 200, such as an integer from 6 to 100, such as an integer from 6 to 40; n is an integer from 4 to 50; The recognition domain of both ends of 10-23 deoxyribozyme forms a Watson-Crick pair with the sequence of the substrate;

N, n, X ₁₅ , X", Xl2, Xii, X X8, Xs, ^4, X2, Xi are as defined in any one of claims 1-13.

17. A kit, kit, or composition, such as a pharmaceutical composition, comprising: i) a 10-23 deoxyribozyme analog according to any one of claims 1 to 14, and optionally ii) a vector or a A pharmaceutically acceptable carrier or excipient, and optionally iii) a product specification or instructions for use.

18. Use of the 10-23 deoxyribozyme analog according to any one of claims 1 to 14, characterized by any one or more of the following:

Use of the 10-23 deoxyribozyme analog in the preparation of a product for genetic research and/or gene therapy, such as a drug, kit, kit; Use of the 10-23 deoxyribozyme analog in the preparation of a product, such as a kit, for use as an endonuclease or as a molecular biology tool; use of a product such as a drug;

Use of the 10-23 deoxyribozyme analog in the preparation of a product, such as a drug, for use as a drug candidate for gene therapy.