A method for linking dual-target nucleic acid molecules and its application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-25
- Publication Date
- 2026-08-14
AI Technical Summary
[0003]临床实践发现，多数疾病的发生发展涉及多基因、多通路协同调控，单靶点干预难以阻断疾病进程
[0015]本发明的有益效果：本发明提供了一种连接两条核酸链的策略方法，两条核酸链之间的连接单元包括一个或一个以上的无碱基核苷酸。两条核酸链之间的连接模式有四种选择：Strand1的5'与Strand2的3'之间、Strand1的5'与Strand2的5'之间、Strand1的3'与Strand2的3'之间、Strand1的3'与Strand2的5'之间。进一步地，无碱基核苷酸连接的两条核酸链，按照碱基互补配对分别与其互补核酸链作用，制备双靶点的寡核酸药物。通过无碱基核苷酸连接的两条核酸链构建双靶点寡核酸药物，连接的核酸链可以作为两条正义链，也可以作为两条反义链，也可以分别作为正义链和反义链。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a novel method for linking dual-target nucleic acid molecules and its applications. Background Technology
[0002] Oligonucleotide drugs bind specifically to target genes, expanding the drug target to the upstream mRNA of pathogenic proteins. By regulating or knocking down target genes, they affect the expression of target genes at the post-transcriptional level, demonstrating clear therapeutic potential in areas such as oncology, viral diseases, and genetic diseases. Oligonucleotide drugs have advantages such as abundant targets, long-lasting efficacy, short development cycles, and high development success rates, providing solutions for the treatment of many intractable diseases.
[0003] Clinical practice has shown that the development of most diseases involves the synergistic regulation of multiple genes and pathways, making it difficult to halt disease progression through single-target intervention. Furthermore, single-target regulation or inhibition can easily lead to mutations in target genes or activation of compensatory pathways, resulting in drug resistance and reduced treatment efficacy.
[0004] To overcome these bottlenecks, dual-target oligonucleotide drugs have emerged. These drugs simultaneously target two genes, achieving a synergistic therapeutic effect of "1+1>2," reducing reliance on single targets and lowering the risk of drug resistance. In recent years, with the maturation of sequence design technology and the optimization of delivery systems, research on dual-target oligonucleotide drugs has gradually moved from basic experiments to clinical applications, becoming a new research focus in the field of nucleic acids.
[0005] In view of this, the present invention is hereby proposed. Summary of the Invention
[0006] The purpose of this invention is to provide a method for linking dual-target nucleic acid molecules and its application, specifically, two nucleic acid chains are linked by a base-free nucleotide unit, and the nucleic acid structure constituting the link includes at least one of the patterns shown in Formulas 1 to 4: ...Equation 1; ...Equation 2; ...Equation 3; ...Equation 4; Strand1 and Strand2 are nucleic acid sequences, including natural or chemically modified DNA or RNA chains; the Linker is a linker unit consisting of one or more non-base nucleotides.
[0007] The linker unit constructed between two nucleic acid strands includes one or more non-basic nucleotides, which are directly or indirectly connected to the 3'-end or 5'-end of the nucleic acid strand. The linker unit includes at least one of the structures shown in Formula 5 or Formula 6. ...Equation 5; ...Equation 6; Among them, R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0008] The present invention prepares coupled nucleic acid chains, wherein the monomers used to introduce baseless nucleotides at the 3' or 5' end of the nucleic acid chain include at least one of the structures shown in Formulas 7 to 10: ...Equation 7; ...Equation 8; ...Equation 9; ...Equation 10; Wherein: PG is a protecting group such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc., respectively; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0009] Preferably, the introduction of a base-free nucleotide at the 3' or 5' end of the coupled nucleic acid strand uses a monomer comprising the structure shown below: , , , ; Wherein: R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0010] Through solid-phase synthesis, a base-free nucleotide phosphoramidite is coupled with nucleotide phosphoramidite or other types of monomers such as phosphoramidite, phosphoramidite chloride, or phosphoramidite and phosphoryl chloride to construct a nucleic acid chain with a designed sequence.
[0011] Solid-phase synthesis processes, including deprotection, coupling, capping, oxidation, or sulfidation, form the synthetic route for constructing oligonucleotide molecules. 1) Deprotection: 3% dichloroacetic acid toluene solution is used to remove O5' or O3' protecting groups, followed by washing with acetonitrile; 2) Coupling: Using 0.25 mol / L 5-ethylthiotetrazole as the activator, according to the sequence design, nucleotide monomers / acetonitrile solutions were fed into the cycle for coupling, followed by rinsing with acetonitrile; 3) Oxidation / Sulfidation: Oxidation was performed using a 0.05 mol / L iodine solution in water / pyridine (v / v, 90 / 10) as the oxidant, followed by rinsing with acetonitrile. Sulfidation was performed using a 3% hydroflavin pyridine solution as the sulfiding agent, followed by rinsing with acetonitrile. 4) Capping: Capping agent A is a 10% acetic anhydride / acetonitrile solution (v / v), and capping agent B is a 1-methylimidazole / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v). Capping agents A and B are used as capping reagents to protect unreacted active groups, followed by rinsing with acetonitrile.
[0012] Following the set sequence, phosphoramide is introduced and steps 1 to 4 are repeated to obtain the oligonucleotide product of the target sequence.
[0013] There are four possible connection modes between the nucleic acid strands (Strand1) and (Strand2): between the 5' of Strand1 and the 3' of Strand2, between the 5' of Strand1 and the 5' of Strand2, between the 3' of Strand1 and the 3' of Strand2, and between the 3' of Strand1 and the 5' of Strand2.
[0014] Oligonucleotides are selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, guide RNAs, transfer RNAs, template DNA, template RNAs, and aptamers.
[0015] The beneficial effects of this invention are as follows: This invention provides a strategy and method for connecting two nucleic acid strands, wherein the connecting unit between the two nucleic acid strands includes one or more base-free nucleotides. There are four possible connection modes between the two nucleic acid strands: between the 5' of Strand1 and the 3' of Strand2, between the 5' of Strand1 and the 5' of Strand2, between the 3' of Strand1 and the 3' of Strand2, and between the 3' of Strand1 and the 5' of Strand2. Furthermore, the two nucleic acid strands connected by base-free nucleotides interact with their complementary nucleic acid strands according to base complementarity pairing to prepare dual-target oligonucleic acid drugs. By constructing dual-target oligonucleic acid drugs using two nucleic acid strands connected by base-free nucleotides, the connected nucleic acid strands can serve as two sense strands, two antisense strands, or both sense and antisense strands, respectively.
[0016] Of course, any product implementing this invention does not necessarily need to achieve all the advantages described above at the same time. Detailed Implementation
[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0018] Example 1
[0019] 0.451 g of S-1-1 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.398 g of solid S-1-2 (yield 61%, purity 99.2%).
[0020] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Among them, R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, amino, aminoacyl, peptide, ester, aromatic, etc.
[0021] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: PG is a protecting group such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc., respectively; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0022] Example 2
[0023] 0.451 g of S-2-1 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.363 g of solid S-2-2 (yield 56%, purity 99.0%).
[0024] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Among them, R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, amino, aminoacyl, peptide, ester, aromatic, etc.
[0025] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: PG is a protecting group such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc., respectively; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0026] Example 3
[0027] 0.406 g of Th-1-1 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.323 g of solid Th-1-2 (yield 53%, purity 99.1%).
[0028] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.
[0029] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: PG is a protecting group such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.; R3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0030] Example 4
[0031] 0.406 g of Th-2-1 (1.0 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry DMF, and 0.056 g of tetrazolium (0.8 mmol, 0.8 eq.) and 0.025 g of N-methylimidazole (0.3 mmol, 0.3 eq.) were added. Under nitrogen protection and at a temperature below 20 °C, 0.5 mL of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.0 mmol, 1.0 eq.) was added. The reaction was allowed to proceed for at least 2 hours, and the reaction was then checked for completion. 25 mL of sodium bicarbonate aqueous solution (3%) was added, and the mixture was extracted twice with 30 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over sodium sulfate, and concentrated. Column chromatography was used to separate 0.302 g of solid Th-2-2 (50% yield, 99.2% purity).
[0032] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.
[0033] Furthermore, similar synthetic procedures are applicable to the following molecular structures: ; Wherein: PG is a protecting group such as triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.; R3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
[0034] Cross-linked polystyrene (PS) was used as the support for solid-phase synthesis. 0.050 g of the modified solid-phase synthesis resin was weighed and added to a solid-phase synthesis tube. 2.0 mL of NMP was added, and the mixture swelled at room temperature for 2 hours, then the solution was drained by filtration. The resin was washed with 3.0 mL of dichloromethane, and this washing process was repeated three times to begin solid-phase synthesis.
[0035] The process conditions are as follows: Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the deprotecting agent for DMTr (or Tr) is a 3% dichloroacetic acid / toluene solution (v / v); the activator is a 0.25 mol / L 5-ethylthiotetrazole / acetonitrile solution; the oxidizing agent is a 0.05 mol / L iodine-water / pyridine (10 / 90, v / v) solution; the sulfiding agent is a 3% hydroflavin / pyridine solution; capping agent A is a 10% acetic anhydride / acetonitrile solution (v / v), and capping agent B is a 1-methylimidazolium / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).
[0036] The sequence of operations for solid-phase synthesis is as follows: 1) Deprotection: 3% dichloroacetic acid toluene solution was used to remove the DMTr protecting group, followed by washing with acetonitrile; 2) Coupling: Using 0.25 mol / L 5-ethylthiotetrazole as the activator, according to the sequence design, nucleotide monomers / acetonitrile solutions were fed into the cycle for coupling, followed by rinsing with acetonitrile; 3) Oxidation / Sulfidation: Oxidation was performed using a 0.05 mol / L iodine solution in water / pyridine (v / v, 90 / 10) as the oxidant, followed by rinsing with acetonitrile. Sulfidation was performed using a 3% hydroflavin pyridine solution as the sulfiding agent, followed by rinsing with acetonitrile. 4) Capping: Capping agent A and capping agent B are used as capping reagents to protect the unreacted active groups, followed by rinsing with acetonitrile.
[0037] Repeat the above operations according to the set sequence to obtain the oligonucleotide product of the target sequence.
[0038] Deprotection involves transferring the solid-phase support loaded with the oligonucleotide product into a reactor, adding concentrated ammonia (28%), controlling the temperature at 45-50 °C for 12 hours of ammonolysis, cooling to room temperature, filtering, washing the solid with a mixture of purified water and ethanol, combining the filtrates, and concentrating under reduced pressure at low temperature to obtain the crude oligonucleotide product with the designed sequence.
[0039] Purification involves dissolving the deprotected crude product in purified water, purifying it using HPLC, collecting the product peak solution, and then desalting, concentrating under reduced pressure, and lyophilizing the product to obtain the final product.
[0040] The above solid-phase synthesis methods are also applicable to monomers protected by Tr, MMTr, and TMTr.
[0041] In examples of solid-phase synthesis, the nucleotide monomers used are represented by the following structures: , , , , , , , , , , , , .
[0042] Example 5
[0043] Monomer U-1 was loaded onto cross-linked polystyrene (PS). Monomers A-1, U-1, C-1, U-1, A-1, G-1, U-1, C-1, U-1, S-1-2, S-1-2, S-1-2, U-1, G-1, U-1, A-1, C-1, U-1, A-1, C-1, G-1, and A-1 were subjected to a cyclic process of de-DMTr, coupling, oxidation, and capping. Following ammonolysis and deprotection treatment, the crude product, concentrated under reduced pressure, was purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain the final product, which was confirmed to be the target sequence nucleic acid molecule 3'-UAUCUAGUCU-5'-S. 12 S 12 S 12 -3'-UGUACUACGA-5', labeled Strand1-1, has the following structure:
[0044] Example 6
[0045] Monomer U-1 was loaded onto cross-linked polystyrene (PS). Monomers A-1, U-1, C-1, U-1, A-1, G-1, U-1, C-1, U-1, S-1-2, DMTr-PEG3-POCE, S-2-2, A-2, G-2, C-2, A-2, U-2, C-2, A-2, U-2, G-2, and U-2 were subjected to a cyclic process of de-DMTr, coupling, oxidation, and capping. Following ammonolysis and deprotection treatment, the crude product, concentrated under reduced pressure, was purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain the final product, which was confirmed to be the target sequence nucleic acid molecule 3'-UAUCUAGUCU-5'-S. 12 -PEG3-S 22 -5'-AGCAUCAUGU -3', labeled Strand1-2, has the following structure:
[0046] Example 7
[0047] Monomer U-1 was loaded onto cross-linked polystyrene (PS). Monomers A-1, U-1, C-1, U-1, A-1, G-1, U-1, C-1, U-1, Th-1-2, Th-1-2, Th-1-2, U-1, G-1, U-1, A-1, C-1, U-1, A-1, C-1, G-1, and A-1 were subjected to a cyclic process of de-DMTr, coupling, oxidation, and capping. Following ammonolysis and deprotection treatment, the crude product, concentrated under reduced pressure, was purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain the final product, which was confirmed to be the target sequence nucleic acid molecule 3'-UAUCUAGUCU-5'-Th. 12 Th 12 Th 12 -3'-UGUACUACGA-5', labeled Strand2-1, has the following structure:
[0048] Example 8
[0049] Monomer U-1 was loaded onto cross-linked polystyrene (PS). Monomers A-1, U-1, C-1, U-1, A-1, G-1, U-1, C-1, U-1, Th-1-2, DMTr-PEG3-POCE, Th-2-2, A-2, G-2, C-2, A-2, U-2, C-2, A-2, U-2, G-2, and U-2 were subjected to a cyclical process of deDMTr removal, coupling, oxidation, and capping. Following ammonolysis and deprotection treatment, the crude product, concentrated under reduced pressure, was purified by HPLC. The main fraction was collected, desalted, concentrated, and lyophilized to obtain the final product, which was confirmed to be the target sequence nucleic acid molecule 3'-UAUCUAGUCU-5'-Th. 12-PEG3-Th 22 -5'-AGCAUCAUGU-3', labeled Strand2-2, has the following structure:
[0050] Example 9
[0051] Monomer A-1 was loaded onto cross-linked polystyrene (PS). Monomers G-1, A-1, C-1, U-1, A-1, G-1, A-1, U-1, and A-1 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection treatment were performed. The crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, desalted, concentrated, and freeze-dried. The product obtained was confirmed to be the nucleic acid molecule 3'-AGACUAGAUA-5' of the target sequence and labeled as Strand0-1.
[0052] Example 10
[0053] Monomer U-1 was loaded onto cross-linked polystyrene (PS). Monomers C-1, G-1, U-1, A-1, G-1, U-1, A-1, C-1, and A-1 were subjected to a cyclical process of de-DMTr, coupling, oxidation, and capping. After the cycle was completed, ammonolysis and deprotection treatment were performed. The crude product concentrated under reduced pressure was separated and purified by HPLC. The main fraction was collected, desalted, concentrated, and freeze-dried. The product obtained was confirmed by detection to be the nucleic acid molecule 3'-UCGUAGUACA-5' of the target sequence and labeled as Strand0-2.
[0054] Example 11
[0055] Nucleic acid molecules Strand1-1, Strand0-1, and Strand0-2 were mixed in equimolar amounts in deionized water, shaken for 10–20 minutes, heated to 80 °C, and then cooled to room temperature (approximately 25 °C) at a rate of 0.5 °C / min. After concentrating the solution, the main fraction was obtained by SEC chromatography and confirmed to be a complementary pairing of Strand1-1 and Strand0-1 / Strand0-2 chains, as shown below:
[0056] Example 12
[0057] Nucleic acid molecules Strand1-2, Strand0-1, and Strand0-2 were mixed in equimolar amounts in deionized water, shaken for 10–20 minutes, heated to 80 °C, and then cooled to room temperature (approximately 25 °C) at a rate of 0.5 °C / min. After concentrating the solution, the main fraction was obtained using SEC chromatography and confirmed to be a complementary pairing of Strand1-2 with Strand0-1 and Strand0-2, as shown below:
[0058] Example 13
[0059] Nucleic acid molecules Strand2-1, Strand0-1, and Strand0-2 were mixed in equimolar amounts in deionized water, shaken for 10–20 minutes, heated to 80 °C, and then cooled to room temperature (approximately 25 °C) at a rate of 0.5 °C / min. After concentrating the solution, the main fraction was obtained using SEC chromatography and confirmed to be a complementary pairing of Strand2-1 and Strand0-1 / Strand0-2 chains, as shown below:
[0060] Example 14
[0061] Nucleic acid molecules Strand2-2, Strand0-1, and Strand0-2 were mixed in equimolar amounts in deionized water, shaken for 10–20 minutes, heated to 80 °C, and then cooled to room temperature (approximately 25 °C) at a rate of 0.5 °C / min. After concentrating the solution, the main fraction was obtained using SEC chromatography and confirmed to be a complementary pairing of Strand2-2 and Strand0-1 / Strand0-2 chains, as shown below:
[0062] This invention provides a method for synthesizing and applying dual-target nucleic acid molecules by constructing a base-free nucleotide linker between two nucleic acid chains. Furthermore, the two base-free nucleotide-linked nucleic acid chains interact with their complementary nucleic acid chains according to base complementarity pairing to prepare dual-target oligonucleic acid drugs.
[0063] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0064] The substances corresponding to the abbreviations in this invention are as follows: DCC: N,N'-Dicyclohexylcarbodiimide; CPG: Controlled pore glass; HPLC: High performance liquid chromatography; SEC: Size exclusion chromatography. Exclusion chromatography); DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; siRNA: Small interfering RNA; P(III): Trivalent phosphorus (phosphine); P(V): Pentavalent phosphorus (phosphine); PS: Polystyrene; F: Fluorine; DIPEA: N,N-Diisopropylethylamine; Ac: Acetyl; Alkyl: Alkyl; Allyl: Allyl; Alloc: Allyloxycarbonyl; Bn: Benzyl; Bz: Benzoyl; DMTr: Dimethoxytriphenylmethyl; PEG: Polyethylene glycol; MMTr: Methoxytriphenylmethyl; Tr: Triphenylmethyl; TMTr: Trimethoxytriphenylmethyl; iBu: 2-Isobutyryl; NPE: 4-Nitrophenylethoxy; Ph: Aromatic group; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; DMSO: Dimethyl sulfoxide; TBAF: Tetrabutylammonium fluoride; THF: Tetrahydrofuran; TBTA: Tris(benzyltriazolylmethyl)amine; A: Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: Hypoxanthine; Xan: Xanthine; m5C: 5-methylcytosine; Ψ: Pseudorabies; m1Ψ: N1-methylpseudorabies; Th: Threonine.
[0065] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the embodiments of apparatus, devices, and non-volatile computer storage media are basically similar to the method embodiments, so the descriptions are relatively simple; relevant parts can be referred to the descriptions of the method embodiments.
[0066] The foregoing has described specific embodiments of this specification. Other embodiments are within the scope of the appended documents. In some cases, the actions or steps described in this application may be performed in a different order than those shown in the embodiments and still achieve the desired results. Furthermore, the described processes do not necessarily require a specific or sequential order to achieve the desired results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.
[0067] The above content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined in this application, they should all fall within the protection scope of the present invention.
Claims
1. A method for linking dual-target nucleic acid molecules and its application, characterized in that, The two nucleic acid chains are linked by a base-free nucleotide unit, and the nucleic acid structure constituting the link includes at least one of the patterns shown in Formulas 1 to 4: ...Equation 1; ...Equation 2; ...Equation 3; ...Equation 4; Strand1 and Strand2 are nucleic acid sequences, including natural or chemically modified DNA or RNA chains; the Linker is a linker unit consisting of one or more non-base nucleotides.
2. The method for linking dual-target nucleic acid molecules according to claim 1 and its application, characterized in that, The linker unit between the two nucleic acid strands includes one or more non-basic nucleotides, which are directly or indirectly linked to the 3'-end or 5'-end of the nucleic acid strand. The linker unit includes at least one of the structures shown in Formula 5 and Formula 6. ...Equation 5; ...Equation 6; Among them, R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc.; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
3. The method for linking dual-target nucleic acid molecules according to any one of claims 1 and 2 and its application, characterized in that, The linking unit between the two nucleic acid strands comprises one or more base-free nucleotides, and the monomers used to introduce base-free nucleotides at the 3' or 5' end of the nucleic acid strands include at least one of the structures shown in Formulas 7 to 10: ...Equation 7; ...Equation 8; ...Equation 9; ...Equation 10; Wherein: PG is a protecting group, including triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, etc.; R1 and R2 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polydiol, aminoacyl, peptide, ester, aromatic, etc., respectively; R3 and R4 are hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, hydroxyl, mercapto, selenyl, azide, cyano, alkoxy, alkoxyalkyl, polydiol, amino, aminoacyl, peptide, ester, aromatic, etc.
4. The method for linking dual-target nucleic acid molecules according to claim 3 and its application, characterized in that, The O5'-hydroxyl group of the nucleic acid chain is coupled with at least one of the monomers shown in Formula 7 or Formula 8 to introduce a baseless nucleotide at the 5'-end of the nucleic acid chain; or one of the O5'-hydroxyl group released by the deprotection of Formula 7 and the O3'-hydroxyl group released by the deprotection of Formula 8 is coupled with a nucleotide phosphoramidite or (thio)phosphoramide to introduce a baseless nucleotide at the 3'-end of the nucleic acid chain.
5. The method for linking dual-target nucleic acid molecules according to claim 3 and its application, characterized in that, The O3'-hydroxyl group of the nucleic acid chain is coupled to at least one of the monomers shown in Formula 9 and Formula 10 to introduce a baseless nucleotide at the 3'-end of the nucleic acid chain; or one of the O3'-hydroxyl group released by the deprotection of Formula 9 and the O2'-hydroxyl group released by the deprotection of Formula 10 is coupled to a nucleotide phosphoramidite or (thio)phosphoramidite to introduce a baseless nucleotide at the 5'-end of the nucleic acid chain.
6. A method for linking dual-target nucleic acid molecules according to any one of claims 1 to 5 and its application, characterized in that, The 3' or 5' linker of the nucleic acid chain is a non-basic nucleotide combined with one or more of the following structures, depending on the physicochemical properties and biological activity requirements: alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester, aromatic, acyl, diacyl, phosphophosphate, thiophosphophosphate, etc.
7. The method for linking dual-target nucleic acid molecules according to claim 1 and its application, characterized in that, The linking unit between the two nucleic acid chains (Strand1) and (Strand2) includes one or more non-base nucleotides, and there are four options for the linking mode of the two nucleic acid chains: between the 5' of Strand1 and the 3' of Strand2, between the 5' of Strand1 and the 5' of Strand2, between the 3' of Strand1 and the 3' of Strand2, and between the 3' of Strand1 and the 5' of Strand2.
8. A method for linking dual-target nucleic acid molecules according to any one of claims 1 to 7 and its application, characterized in that, Oligonucleotide drugs targeting two bases can be prepared by binding their complementary nucleic acid chains to two base-complementary chains.
9. A method for linking dual-target nucleic acid molecules according to any one of claims 1 to 8 and its application, characterized in that, Dual-target oligonucleotide drugs can be constructed by linking two nucleic acid strands without base nucleotides. The linked nucleic acid strands can serve as two sense strands, two antisense strands, or as sense strands and antisense strands respectively.
10. The method for linking dual-target nucleic acid molecules according to claim 1 and its application, characterized in that, The oligonucleotide is selected from any one or a combination of at least two of small interfering nucleotides, antisense oligonucleotides, microRNAs, small activating RNAs, guide RNAs, transfer RNAs, template DNA, template RNAs, and aptamers.