1. A method of gene therapy by inducing proton transfer of a pyrimidine or purine to alter its pairing properties, comprising: the method comprises the following steps:

step S100: constructing a molecular structure theoretical model of classical pyrimidine or purine and a pyrimidine purine compound;

step S200: forming a plurality of new pyrimidine or purine analogue molecular structures by replacing atoms or groups on a classical pyrimidine or purine;

step S300: calculating a plurality of physicochemical parameters of each new molecule which interacts with the pyrimidine or purine and undergoes proton transfer;

step S400: evaluating each parameter, and screening out candidate molecules capable of inducing pyrimidine or purine to generate proton transfer under physiological conditions;

s500: the candidate molecules are used as core raw materials to be synthesized into drug molecules for single base mutant gene therapy or other purposes.

2. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S100, the site where pyrimidine or purine binds to the sugar ring is replaced with a methyl group or a hydrogen group.

3. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S100, the structure of single molecules and complexes is optimized by M06X or B3 LYP.

4. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S200, in order to enhance the ability of the N atom in the base pair hydrogen bond to acquire a proton, the atom or group upstream and downstream of the N atom may be replaced with a less electronegative atom or group.

5. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S200, in order to make it easier for the amino group in the base pair hydrogen bond to provide a proton, the amino group may be replaced with a hydroxyl group or a thiol group.

6. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in the step S300, the proton transfer reaction between the pyrimidine or purine analog and its counterpart pyrimidine or purine is simulated by using the density functional theory, M062X or B3LYP method, and def2sv or 6-31+ + g (d, p) and other groups.

7. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S300, all calculations are performed under water solvation conditions of T-310.15K and p-1 atm.

8. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in step S400, the difference between the energy (Delta E) after intramolecular proton transfer and the energy (Delta E) before the transfer of the designed molecule is used as a criterion for evaluating the stability of the molecule, the chemical reaction equilibrium ratio (Keq) of proton transfer of pyrimidine or purine induced by the designed molecule is used as a criterion for evaluating the ability of proton transfer induced by the pyrimidine or purine induced molecule, and molecules having Delta E >0 and Keq >1 are selected as candidate molecules.

9. A method of gene therapy according to claim 1 by inducing proton transfer of pyrimidines or purines to alter their pairing properties, wherein: in the step S500, the candidate molecules are used for synthesizing the drug, and the matching property of the drug is changed by inducing the pyrimidine or purine to generate proton transfer for gene therapy or other purposes.

Background

Gene therapy is the structural or functional repair of mutant genes by various methods and strategies to achieve the goal of treating disease. The main strategies of current gene therapy include gene addition, gene replacement, RNA/DNA chimeric oligonucleotide-mediated site-directed repair techniques, the use of complementary binding of antisense nucleic acids to target genes, suppression of gene expression at the transcriptional and translational levels, introduction of small segments of RNA into target cells to regulate mRNA splicing, intervention at the RNA level to cause exon skipping or cause mRNA degradation, repair of DNA sequences at the site of mutation by gene editing techniques, and the like. Human genetic diseases of nearly 2/3 are caused by single base variation, and thus the single base editing technology is a hot spot technology for gene therapy research.

The existing single base editing technology combines a CRISPR/Cas system and deaminase to hydrolyze amino on pyrimidine or purine into carbonyl so as to realize the editing of mutant base, but the system needs to introduce an enzyme system into cells or cell nuclei, has a large system and has the defects of off-target, additional deletion or insertion, limited vector capacity, triggering innate immune reaction and the like, so that the CRISPR/Cas system single base editing technology cannot be effectively applied to clinical treatment at present.

Disclosure of Invention

The invention provides a method for carrying out gene therapy by inducing pyrimidine or purine to generate proton transfer to change pairing property of pyrimidine or purine, and aims to solve the problems that a CRISPR/Cas single base editing enzyme complex system is too large, the gene therapy process is complex and difficult, and various side effects such as immunoreaction and the like are generated, so that the CRISPR/Cas single base editing technology cannot be effectively applied to clinical therapy.

The present invention is achieved by a method for gene therapy by inducing proton transfer of pyrimidine or purine to change its pairing property, comprising the steps of:

step S100: constructing a molecular structure theoretical model of classical pyrimidine or purine and a pyrimidine purine compound;

step S200: forming a plurality of new pyrimidine or purine analogue molecular structures by replacing atoms or groups on a classical pyrimidine or purine;

step S300: calculating a plurality of physicochemical parameters of each new molecule which interacts with the pyrimidine or purine and undergoes proton transfer;

step S400: evaluating each parameter, and screening out candidate molecules capable of inducing pyrimidine or purine to generate proton transfer under physiological conditions;

s500: the candidate molecules are used as core raw materials to be synthesized into drug molecules for single base mutant gene therapy or other purposes.

Preferably, in step S100, the site where the pyrimidine or purine binds to the sugar ring is replaced with a methyl group or a hydrogen group.

Preferably, in step S100, the structure of the single molecule and the complex is optimized by using methods such as M06X or B3 LYP.

Preferably, in step S200, in order to enhance the ability of the base pair to take out protons from the N atom in the hydrogen bond, the upstream and downstream atoms or groups thereof may be replaced with atoms or groups having a weaker electronegativity.

Preferably, in step S200, in order to make it easier for the amino group in the base pair hydrogen bond to provide a proton, the amino group may be replaced with a hydroxyl group or a thiol group.

Preferably, in step S300, the proton transfer reaction between the pyrimidine or purine analog and its counterpart pyrimidine or purine is simulated using density functional theory, M062X or B3LYP method, and def2sv or 6-31+ + g (d, p) group.

Preferably, in step S300, all calculations are performed under water solvation conditions of T-310.15K and p-1 atm.

Preferably, in step S400, the difference between the energy (Δ E) after intramolecular proton transfer and the energy (Δ E) before proton transfer of the designed molecule is used as a criterion for evaluating the stability of the molecule, the chemical reaction equilibrium ratio (Keq) of proton transfer of pyrimidine or purine induced by the designed molecule is used as a criterion for evaluating the ability of proton transfer induced by the designed molecule, and molecules having Δ E >0 and Keq >1 are selected as candidate molecules.

Preferably, in step S500, the drug is synthesized from the candidate molecule, and the pairing property of the drug is changed by inducing the proton generation of pyrimidine or purine to perform gene therapy or other uses.

Compared with the prior art, the invention has the beneficial effects that: the invention relates to a method for carrying out gene therapy by inducing pyrimidine or purine to generate proton transfer and changing the pairing property thereof, which is characterized in that candidate molecules with specific structures are theoretically modeled, designed and calculated, so that the candidate molecules have the effects of inducing thymine, cytosine, uracil, adenine and guanine to generate proton transfer and changing the pairing property under physiological conditions, and thus, the method and the thought for gene therapy are realized.

Drawings

FIG. 1 is a schematic diagram of the process steps of the present invention;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Referring to FIG. 1, the present invention provides a method for gene therapy by inducing proton transfer of pyrimidine or purine to change its pairing property, comprising the steps of:

step S100: constructing a molecular structure theoretical model of classical pyrimidine or purine and a pyrimidine purine compound;

the molecular structures of thymine, adenine and thymine-adenine complexes were constructed by Gaussian (Gaussian) or the like software, and the sites in thymine and adenine that bind to the sugar ring were replaced with methyl or hydrogen groups in order to mimic the minimal structure of classical base pairs. The density functional theory and the M06X or B3LYP method are adopted to carry out structure optimization on the single molecules and the compound, and the optimal structure is obtained. The results are shown in the following figures:

step S200: forming a plurality of new pyrimidine or purine analogue molecular structures by replacing atoms or groups on a classical pyrimidine or purine;

in order to enhance the capability of adenine to acquire protons at the N1 position, a hydrogen group at the C2 position can be replaced by an amino group, a carbon atom at the C2 position can be replaced by a boron atom, and a nitrogen atom at the N3 position can be replaced by a carbon atom; in order to make N6 provide proton more easily, the amino group at C6 position can be replaced by hydroxyl or sulfhydryl, and other atom or group replacement or modification can be carried out. The adenine analog structure formed by the replacement of atoms or groups in the following moieties:

step S300: a number of physicochemical parameters were calculated for each new molecule to interact with the pyrimidine or purine and for proton transfer to occur.

The proton transfer reaction between adenine analogs and thymine was simulated using density functional theory, the M062X or B3LYP method, and the def2sv or 6-31+ + g (d, p) group set. To improve the accuracy of the energy and structure calculations, D3 dispersion correction was used. To mimic the physiological environment in which a DNA molecule is located, all calculations were performed under water solvation conditions of T-310.15K (37 ℃) and p-1 atm. All calculations were performed using software such as the gaussian package. On the theoretical level, important parameters for evaluating proton transfer, such as structure optimization, total molecular energy, transition state energy barrier of proton transfer, chemical reaction rate, reaction equilibrium constant and the like, are calculated for all molecular monomers and compounds. The following table shows the calculated values of the parameters for the molecular interstitial transfer of the partial adenine analog and thymine complex.

Aan

△E

△G≠ f

△G≠ r

kf(s-1)

kr(s-1)

Keq

Aa1

—

8.79

-1.77

4.11×106

1.14×1014

3.61×10-8

Aa2

11.07

4.93

-0.61

2.16×109

1.74×1013

1.24×10-4

Aa3

-0.43

-0.65

1.03

1.86×1013

1.21×1012

1.54×101

Aa4

9.48

3.32

1.21

2.95×1010

9.07×1011

3.25×10-2

Aa5

11.97

3.01

1.04

4.88×1010

1.20×1012

4.07×10-2

Aa6

1.79

-0.46

0.34

1.36×1013

3.72×1012

3.66×100

Aa7

-2.35

0.33/-1.17/0.42

-1.46/-2.96/-1.37

3.27×1012

5.97×1013

5.48×10-2

Aa8

-2.05

0.11

4.74

5.41×1012

2.94×109

1.84×103

Aa9

-8.55

-0.59/-3.61/-3.35

1.55/-1.47/-1.21

1.68×1013

5.22×1011

3.22×101

Aa10

-6.16

-0.52

5.02

1.51×1013

1.87×109

8.07×103

Aa11

-1.01

1.47

-0.38

5.95×1011

1.20×1013

4.96×10-2

Aa12

-0.94

2.44

0.73

1.23×1011

1.98×1012

6.21×10-2

Aa13

-0.93

-0.24

2.62

9.54×1012

9.19×1010

1.04×102

Aa14

-0.63

1.94

0.26

2.77×1011

4.24×1012

6.53×10-2

Aa15

-0.54

1.73

1.73

3.90×1011

3.90×1011

1.00×100

Aa16

-0.72

-0.60

3.60

1.17×1013

1.87×1010

9.14×102

Aa17

-0.96

-0.52

6.30

1.50×1013

2.34×108

4.41×104

Aa18

-0.86

-1.23

6.10

4.74×1013

3.23×108

1.47×105

Aa19

-0.34

-1.16

9.72

4.25×1013

9.09×105

4.68×107

Aa20

-0.13

-0.48

8.18

1.41×1013

1.11×107

1.27×106

Note: energy difference (Delta E) between the product and the reactant after intramolecular proton transfer; PT Forward reaction energy Barrier (Δ G)^≠ _f) (ii) a PT reverse reaction barrier (Δ G)^≠ _r) (ii) a PT Forward reaction Rate (k)_f) (ii) a Reverse reactionA ratio (kr); PT reaction equilibrium constant (Keq); all energy barrier values are in kcal/mol.

Step S400: evaluating each parameter, and screening out candidate molecules capable of inducing pyrimidine or purine to generate proton transfer under physiological conditions.

And selecting molecules which are stable and are easy to induce proton transfer as candidate molecules according to the calculated parameter values. The difference between the energy (Delta E) after intramolecular proton transfer and that before the transfer of the designed molecule was used as a criterion for evaluating the stability of the molecule, the chemical reaction equilibrium ratio (Keq) of proton transfer of pyrimidine or purine induced by the designed molecule was used as a criterion for evaluating the ability of proton transfer induced by the designed molecule, and molecules having Delta E >0 and Keq >1 were selected as candidate molecules. For example, Aa6 shows that the molecular structure is stable and Keq is greater than 1, indicating that thymine proton transfer is likely to be induced. Therefore, Aa6 molecule can be used as candidate molecule.

S500: the candidate molecules are used as core raw materials to be synthesized into drug molecules for single base mutant gene therapy or other purposes.

Aa6 is synthesized into phosphoramidite monomer, and is further synthesized into a specific ssDNA sequence or RNA sequence, through combination of ssDNA or RNA and target gene or RNA, Aa6 molecule is paired with thymine of a target site and undergoes proton transfer reaction, so that the thymine pairing property of the target site is changed, and the cytosine pairing property is presented, thereby realizing the purposes of gene therapy or other purposes.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.