WO2007012285A1

WO2007012285A1 - Viral infection resistent single strand deoxynucleosides

Info

Publication number: WO2007012285A1
Application number: PCT/CN2006/001891
Authority: WO
Inventors: Li-Ying Wang; Mu-Sheng Bao; Yong-Li Yu
Original assignee: Changchun Huapu Biotechnology Co., Ltd.
Priority date: 2005-07-28
Filing date: 2006-07-28
Publication date: 2007-02-01

Abstract

Single strand deoxynucleosides useful for viral infective disease treatment. These single strand deoxynucleosides could stimulate the human cells to produce antiviral substance, which could inhibit the replication of the virus. Also the composition containing the deoxynucleosides, the kit and the use for viral infective disease treatment thereof is disclosed.

Description

Anti-viral infection single-stranded deoxynucleotides

Field of invention

The present invention relates to a single-stranded deoxynucleotide which has a therapeutic effect on viral infectious diseases.

(allophone, hereinafter referred to as oligo). These single-stranded deoxynucleotides can stimulate human cells to produce antiviral substances that inhibit the replication of viruses including, but not limited to, hepatitis B virus, HIV, and thus viral infectious diseases including but not limited to Hepatitis B caused by hepatitis B virus and diseases caused by immunodeficiency virus such as HIV infection have therapeutic effects. Background of the invention

A virus is a type of non-cellular microorganism that is small in size and relatively simple in structure and can only proliferate in living cells. The structure of the virus includes nucleic acids, capsids and envelopes. The nucleic acid of a virus, the genome of a virus, is the core of the virus and determines the inheritance, variation and replication of the virus. The capsid and membrane act to protect the core of the virus and are antigenic. According to the type of viral nucleic acid, the virus is classified into a DNA virus and an RNA virus. DNA viruses include double-stranded DNA viruses and single-stranded DNA viruses; RNA viruses include single-stranded RNA viruses, single-negative-strand RNA viruses and double-stranded RNA viruses, and retroviruses. DNA viruses include, but are not limited to, hepatitis B virus, TTV virus, adenovirus, papilloma virus, herpes zoster virus, variola virus, and vaccinia virus. RNA viruses include, but are not limited to, influenza virus, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus And immunodeficiency viruses such as HIV.

As of December 2003, 40 million people worldwide were infected with HIV (Onyinye I. Iweala. HIV diagnostic tests: an overview. Contraception, 2004, (70): 141 - 147.). AIDS, also known as Acquired Immunodeficiency Syndrome (AIDS), is a contagious disease caused by human immunodeficiency virus (HIV).

The human immunodeficiency virus has a circular shape with a diameter of 100nm-120nm. Under the electron microscope, a dense conical core is visible, containing viral RNA molecules and enzymes (reverse transcriptase, integrase and protease), and the outer membrane of the virus is double-layered. A protein membrane, in which gpl20 protein and gp41 protein are embedded, which constitute the spike and transmembrane proteins of the virus, respectively. The inner surface of the capsule is a capsid composed of P17 protein. The viral center consists of a core protein (P24) and viral RNA.

The HIV genome is about 9. 2 kb in length and contains three structural genes of gag, pol and env and at least six regulatory genes (Tat Rev, Nef, Vif, VPU, Vpr) and each of the 5' and 3' ends of the genome. End repeats. Gag The gene encodes viral core proteins such as p24, p9, p6, pl7, etc.; the pol gene encodes a variety of enzymes, including reverse transcriptase; the er gene encodes a large viral precursor glycoprotein g _P 160, gpl60 after translation Degraded to gpl20 and gp41 (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, p. 295-305.).

In the weeks after HIV infection, viremia caused by HIV occurs in infected people. At the same time, HIV-specific immune responses occur in most infected people, and the result is a reduction in HIV viremia. After that, a typical infected person will enter a stable, asymptomatic period that can last for years or longer. In this period, the patient's HIV viremia was lighter and there was no significant reduction in the number of CD4+ cells, mainly due to HIV replication and clearance, and the destruction and complementation of infected CD4+ cells was in a dynamic equilibrium (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, P. 295 - 305. Over time, if the level of HIV in the peripheral blood continues to rise, HIV-infected people will develop AIDS.

The development of anti-HIV drugs and preparations is the key to the treatment of AIDS, and the evaluation of the therapeutic effect of anti-AIDS drugs is the focus of anti-AIDS drugs. The detection of HIV infection mainly includes detection of viral nucleic acid, p24 antigen and anti-HIV antibody. Peripheral blood HIV levels are important indicators of disease progression and treatment outcomes in HIV-infected individuals. Quantitative detection of HIV p24 antigen and HIV RNA is an important method for detecting HIV in human peripheral blood (STANLEY A. SCHWARTZ AND MADHAVAN PN NAIR. Current Concepts in Human Immunodeficiency Virus Infection and AIDS. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1999, p. 295 - 305. ).

P24 is the core protein of HIV encoded by the structural gene gag of the HIV virus (Iweala 01. HIV diagnostic tests: an overview. Contraception 2004, Aug; 70 (2): 141-7.). Studies have shown that antiviral drugs can lower the level of p24 antigen in the blood. Therefore, the level of p24 antigen in the blood is a very useful marker for evaluating the antiviral effect of antiviral agents (Spector, SA, et al. The antiviral effect). Of zidovudine and ribavirin in clinical trials and the use of p24 antigen levels as a virologic marker. J. Infect. Ms. 1989,

159 : 822 - 828.; Burgisser, P. , et al. Performance of five different assays for the quantification of viral load in persons infected with various subtypes of HIV — 1. J. Acquir. Immune Defic. Syndr. 2000, 23 : 138 - 144). P2 can occur in serum isolated from HIV-infected individuals, and the p24 antigen can be detected in approximately 20% of asymptomatic HIV-infected individuals and 40-50% of AIDS patients using the enzyme immunoassay kit. HIV-infected cells cultured in vitro can be released into the supernatant _P 24, it is an indication of the release of p24 HIV replication, and the amount released is proportional to the replication of the virus. HIV replication is inhibited and the level of p24 is reduced.

Viral hepatitis is an infectious liver inflammatory disease caused by hepatitis virus (H-printed atitis virus). There are seven types of viruses that cause viral hepatitis, namely Hepatitis A virus (HAV), Hepatitis B virus (HB), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatitis E virus (Hepatitis E virus), Hepatitis G virus (HGV), and sputum (transfusion-transmitted viral hepatitis virus), which can Causes A, B, C, D, E, G and TTV hepatitis.

Hepatitis A virus (HAV) is a spherical, non-enveloped, single-stranded RNA virus. Hepatitis A is referred to as Hepatitis A, an intestinal infectious disease caused by HAV (Atmar, RL, et al. Detection of enteric viruses in oyster by using the polymerase chain reaction. Appl. Environ. Microbiol. 1993, 59 : 631 - 635. ). Symptoms of acute hepatitis A include fever, loss of appetite, weakness, hepatomegaly, and abnormal liver function. Only a small number of patients with acute hepatitis A have jaundice. Hepatitis A virus generally does not turn into a chronic and pathogen-carrying state. Hepatitis A patients are most contagious from the latent stage to the 10 days after onset. Hepatitis A usually occurs in children and adolescents and is rare in adults. The general incubation period is 2 to 6 weeks. The winter and spring seasons are the peak of the onset of hepatitis A (JENNIFER A. CUTHBERT et al. Hepatitis A: Old and New. CLINICAL MICROBIOLOGY REVIEWS, Jan. 2001, p. 38-58).

Hepatitis C virus (HCV) is a genus of flavivirus, a single-stranded positive-strand RNA virus, having a diameter of about 50 to 60 nm and having a lipid envelope. Hepatitis C is mainly transmitted through blood and blood products. According to the data, 90% of hepatitis infected by blood transfusion is hepatitis C (JANICE M. MATTHEWS-GREER, et al. Comparison of Hepatitis C Viral Loads in Patients with or without Human Immunodeficiency Virus. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 2001, p. 690 - 694; Luo Kangxian et al., Hepatitis C virus subtypes and their possible implications. Journal of Clinical Hepatology, 1994, 10 (2): 61-62. > Hepatitis C and cirrhosis and hepatocytes The occurrence of cancer is closely related (NIZAR N. ZEIN. Clinical Significance of Hepatitis C Virus Genotypes. CLINICAL MICROBIOLOGY REVIEWS, 2000, p. 223 - 235; Yang Yongping, cloning the hepatitis C virus genome C region gene from Chinese patients and Expression in E. coli. Acta Zoologica Sinica, 1994, 10 (2): 109-117).

Hepatitis D virus (HDV) is a defective virus whose nucleic acid is a single negative stranded circular RNA (Theo Heller and Jay H. Hoofnagle. Denying the wolf access to sheep's clothing. J. Clin. Invest. 2003, 112:319-321 ). As a defective virus, HDV requires the help of a hepadnavirus (such as HBV) to complete its life cycle. Hepatitis D virus is transmitted in a similar manner to hepatitis B virus, with vertical transmission, sexual contact and body fluid transmission as the main modes of transmission (Rizzetto, M., et al. 1980. Transmission of the hepatitis B virus-associated delta antigen To chimpanzees. J. Infect. Dis. 141 : 590 - 602). Infection with HDV can aggravate the clinical symptoms of HBV-infected patients (Hadler, SC et al. Delta virus infection and severe hepatitis. An epidemic in the Yucpa Indians of Venezuela. Ann. Intern. Med. 1984. 100: 339-344.).

Hepatitis E virus (HEV) is a spherical, non-enveloped virus with a diameter of 32–34 nm. Its nucleic acid is a single positive-stranded RNA containing three reading frames (Reyes, GR, et al. Isolation of a cDNA from the virus responsible for Enterically transmitted non - A, non-B hepatitis. Science. 1990: 247 : 1335 - 1339; Reyes, GR , et al. Hepatitis E virus (HEV): the novel agent responsible for enterically transmitted non- A, non - B hepatitis .

Gastroenterol, jpn. 1991, 26 (Suppl. 3): 142 - 147. ). Hepatitis E, like hepatitis A, is transmitted through the faecal-oral route, because the water source is contaminated and causes large outbreaks and epidemics. The pathological damage caused by hepatitis E is more obvious than that of hepatitis A, and it recovers slowly.

Hepatitis G virus (HGV/GBV-C) is a enveloped, single-stranded positive-strand RNA virus of the Flaviviridae family that replicates mainly in the liver, mainly through blood products, intravenous drugs, sexual contact, and vertical transmission of mother and child. Hepatitis G virus can cause acute and chronic hepatitis, and is even associated with the occurrence of liver cancer and cirrhosis (Schmidt, B., K. Korn, and B. Fleckenstein. Molecular evidence for transmission of hepatitis G virus by blood transfusion. Lancet. 1996 , 347 : 909; Iban~ ez, A. , M. Gime' nez-Barcons, et al. Prevalence and genotypes of GB virus C/hepatitis G virus (GBV-C/HGV) and hepatitis C virus (HCV) among human Immunodeficiency virus (HIV) infected patients : evidence of GBV-C/HGV sexual transmission, j. Med. Virol. 1998, 55 : 293 - 299; Wang Yongmei, research status of hepatitis G. Journal of Preventive Medicine Information, 2001, 17 ( 4): 257-258; Sun Keer et al., Recent research on hepatitis G virus. Foreign Medical Virology, 1997, 4 (2): 33-36.

Transfusion transmitted virus (TTV) is a non-enveloped, single-stranded awakening virus (C.-L. lin, et al. Fecal Excretion of a Novel Human Circovirus, TT Virus, in Healthy Children. Clinical and diagnostic laboratory immunology, 2000, p. 960 - 963 ). The genome of the TTV virus is a circular DNA ( Isa k. Mushahwar, et al. Molecular and biophysical characterization of TT virus: Evidence for a new virus family infecting humans. Proc. Natl. Acad. Sci. USA Vol. 96, pp 3177 - 3182, March 1999 . Microbiology) . TTV virus can be transmitted through sexual contact, vertical transmission, etc. Claudia Fornai, et al. High Prevalence of TT Virus (TTV) and TTV-Like Minivirus in Cervical Swabs. JOURNAL OF CLINICAL MICROBIOLOGY , 2001, p. 2022 - 2024 ).

Hepatitis B virus (HBV) is a DNA virus of the Department of Hepatic DNA. The complete HBV consists of an envelope and a core. Hepatitis B surface antigen (HBsAg) is the main component of the envelope. HBsAg is synthesized in hepatocytes and released into the blood circulation in a large amount, which is not contagious in itself. The core contains incompletely circular double-stranded DNA, DNA polymerase, core antigen (HBcAg) and e antigen (HBeAg), which are infectious. Hepatitis B virus is a inflammatory disease of the liver caused by hepatitis B virus that is a serious threat to human health. As of 2004, 400 million people worldwide have been infected with the hepatitis B virus (Lin KW, Kirchner JT. Hepati ti s B. Am Fam Physician, 2004 Jan 1, 69 (1): 75-82.), Among them, Asians make up the majority.

In order to develop a drug that has a therapeutic effect on hepatitis B virus infection at the cellular level, Mary ann sells et al. constructed HepG2.2.15 cells. HepG2. 2. 15 cells are hepatoma cells (HepG2 cells) stably transfected with the HBV genome, which are capable of stably secreting HBsAg, HBeAg and Dane particles. HepG2 cells can maintain many functions of normal hepatocytes during long-term subculture, such as ensuring normal replication of HBV, expressing multiple receptors such as interferon and Tol l-like receptors (Volker Mersch-Sundermann. Use of a human-derived l iver cel ll ine for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology, 2004, 198 : 329 - 340; Mary ann sells, Mei-long chen, and George acs. Of hepatitis B virus particles in Hep G2 cel ls transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci, USA, 1987, 84: 1005-1009). Studies have shown that antiviral substances, including but not limited to interferon, bind to the corresponding receptors of HepG2.2.15 cells, thereby exerting an antiviral effect. Interferon induces the production of interferon regulatory factor 1 (IFN-regulated factor 1, IFN-1), RNase L by complex intracellular signal transduction after acting on the corresponding receptor on the surface of He _P G2. Expression of (RNase L) and double-stranded RNA activated protein kinase (PKR) to exert antiviral activity including but not limited to HBV (Bachmaier, K. et al. IiNOS expression) And nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1. Circula tion, 1997, 96 : 585-591; Pitha, PM , et al. Role of the interferon regulatory factors in virus-mediated signaling and Regulation of cell growth. Biochimie, 1998, 80: 651-658; Zhou, A. et al. Expression cloning of 2-5A-RNAase: uniquely regulated mediator of interferon action. Cell, 1993, 72: 753-765). Using HepG2. 2. 15 cells for in vitro experiments, the activity of antiviral substances can be expressed as a decrease in the content of HBsAg, HBeAg and HBV nucleic acids in the cell culture supernatant. Therefore, HepG2.2.15 cells are a good cell model for studying anti-HBV drugs and preparations. In the present invention, we used a single-stranded deoxynucleotide-induced human peripheral blood mononuclear cell (PBMC) culture supernatant and He _P G2.2.15 cells, according to HBsAg, HbeAg and HBV detected in the supernatant. The amount of nucleic acid is used to determine the biological effect of a single-stranded deoxynucleotide against hepatitis virus. Summary of the invention

In the context of the present invention, the terms used generally have the meanings as commonly understood by one of ordinary skill in the art, unless otherwise indicated.

In one aspect, the invention relates to a synthetic single-stranded deoxynucleotide for use in antiviral infection having the structural formula shown in 1-5 below:

1. Synthetic single-stranded deoxynucleotides conforming to the formula (G)n(L)ii XlX2CGYlY2(M)n (G)N (X1=A, T, G; X2=A, T; Y1=A, T; Y2 = A, T, C; L, M = A, T, C, G; n is 0-6, preferably,

1. 5 ' -ggggTCgTTCgTCgTTgggggg-3 '

2. 5 ' -ggggATAACgTTgCgggggg-3 '

3. 5 '-ggggTgCAACgTTCAgggggg-3 '

4. 5 ' -ggggTCCTACgTAggAgggggg-3 '

5. 5 '-ggggTCCATgACgTTCCTgAAgggggg-3 '

6. 5 '-gggggACgTCgCCggggggg-3 '

7. 5'-ggATCCgTACgCATgggggg-3'

8. 5'-gggggAATCgATTCgggggg-3'

9. 5'-gggATgCATCgATgCATCgggggg-3'

10. 5'-ggT _g CgACgTCgCAgggggg-3' 1 1. 5'-gggAC _g TACgTCgggggg-3'

12. 5'-gggggATCgACgTCgATCgggggg-3,

13. 5'-ggCgATCgATCgATCggggggg-3'

14. 5'-ggggTCgATCgATCgAgggggg-3'

15. 5'-ggTCgCgATCgCgAgggggg-3'

16. 5'-ggGGTCAACGTTGAgggggG-3'

17. 5'-gTCgTTTTCgTCgACgAATTgggggggg-3'

18. 5'-gTCgTTATCgTTTTTTCgTAgggggg-3'

19. 5'-ggCgTTAACgACgggggg-3'

20. 5'-gTCggCACgCgACgggggg-3'

21. 5'-ggTgCgACgTCgCAgggggg-3'

22. 5'-gTCTATTTTgTACgTACgTgggg-3'

23. 5'-gACgTCgACgTCgACgTCAggggg-3'

24. 5'-ggggTCgATCgTTgCTAgCgggggg-3'

25. 5'-gggggACgTTATCgTATTggggggg-3'

26. 5'-ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3'

27. 5 ' -ACgATCgATCgATCgggggg-3 '

28. 5'-AgACgTCTAACgTCggggg-3'

29. 5'-ggggTgCTggCCgTCgTTgggggg-3'

30. 5'-ggggTCgTTgCCgTCgggggg-3'

31. 5'-ACCggTATCgATgCCggTgggggg-3'

32. 5'-TTCgTTgCATCgATgCATCgTTgggggg-3'

2. Synthetic single-stranded deoxynucleotides conforming to the formula (G)n(L)nCG(XY)nCG(M)n(G)n (XA, T; Y=A, T; L, M=A , T, C, G; n is 0-6, preferably,

33. 5'-ggggACgATACgTCggggggg-3'

34. 5'-ggggACgATATCgATgggggg-3'

35. 5'-ggACgATCgATCgTgggggg-3'

36. 5'-TCggggACgATCgTCgg _g ggg-3'

37. 5'-g _ggg gATC _g ATATCgATCgggg _g g-3' 'B 9 οε

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168T00/900ZM3/X3d Off 00Ζ OAV 182.5'-TCgTCgTTTTgCTgCgTCgTT-3'

183.5'-TCgAgCgTTTTCgCTCgAATT-3'

4. A synthetic single-stranded deoxynucleotide comprising a sequence formula of TTCGTCG, preferably,

184.5'-TTCgTCgTTTgATCgATgTTCgTTgggggg-3'

185.5'-TTCgTCgTTgTgATCgATgggggg-3'-3'

186.5'-TATCgATgTTTTCgTCgTCgTTgggggg-3 '

187.5'-TCgACTTTCgTCgTTCTgTT-3'

188.5'-TCgTCgTTTCgTCgTTCTC-3'

189.5'-TCgACgTTCgTCgTTCgTCgTTC-3'

190.5'-TCgTCgTTTTCgTCgTTTTCgTCgTT-3 '

More preferably, the synthetic single-stranded deoxynucleotides of the invention have the sequence shown below:

Oligol Seq No 89 : 5'-TCgTCgggTgCgATCgCAgggggg-3'

01igo2 seq No 92 5'-TCgTCgCAgAACgTTCTgggggg-3'

01igo4 seq No 95 5'-TCgTATgCATCgATgCATAgggAgg-3'

Oligo3 seq No 174 5 '-TCgTgCgACgTCgC AgATgAT-3 '

Seq No 164 5, -TCgTCggTCTTTCgAAATT-3 '

01igo6 seq No 36 5'-TCggggACgATCgTCgggggg-3'

01igo7 seq No 172 5 '-TCgACgTTCgTCgTTCgTCgTTC-3 '

01igo8 seq No 173 5, -TCgTCgACgTCgTTCgTTCTC-3 '

01igo9 seq No 98 5'-TCggACgATCgTCgggggg-3'

OligolO seq No 99 5'-TCgAgCgATCgCTCgAgggggg-3'

Oligol 1 seq No 107 5'-TCgTCgggTgCgACgTCgCAgggggg-3'

Oligol 2 seq No 170 5'-TCgTCgggTgCgACgTCgCA-3'

Oligol 3 seq No 102 5'-TCgTCgggTgCgACgTCgCAg-3'

Oligol 4 seq No 103 5'-TCgTCgggTgCgACgTCgCAgg-3'

Oligol 5 seq No 104 5'-TCgTCgggTgCgACgTCgCAggg-3'

Oligol 6 seq No 105 5'-TCgTCgggTgCgACgTCgCAgggg-3'

Oligol 7 seq No 106 5'-TCgTCgggTgCgACgTCgCAggggg-3'

Oligol 8 seq No 3 5'-ggggTgCAACgTTCAgggggg-3' 01igol9 seq No 1 : 5'-ggggTCgTTCgTCgTTgggggg-3'

Oligo20 seq No 66: 5'-gggggCgTCgTTTTCgTCgACgAATT-3'

One of ordinary skill in the art, based on the teachings of the specification, in conjunction with common general knowledge in the art, is well established that the above-described synthetic single-chain deoxynucleotides can be used to achieve the objectives of the present invention.

The artificially synthesized single-chain deoxynucleotides of the present invention can be produced by a known method, for example, by a solid phase phosphoramidite triester method.

The single-stranded deoxynucleotides described in the present invention may be modified by various chemicals. These chemical modifications include, but are not limited to, phosphate backbone modifications, sugar ring modifications, and base modifications. The modification of the phosphate backbone can be a thio modification. Thio modification refers to the substitution of a non-bridged oxygen atom in a phosphodiester bond by a sulfur atom in a nucleotide fragment (Ekambar R. Kandimalla et al. Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2 -5 - Ribo - and 3 - 5 -deoxyribonucleotide segments. Nucleic Acids Research, 1997, 25 ( 2 ) : 370 - 378 ) , this substitution may occur in part or all of the phosphodiester bond of the deoxynucleotide. Modification of the phosphate backbone can be accomplished by the formation of other types of phosphate linkages including, but not limited to, methyl phosphates, selenophosphates, boryl phosphates, and phosphorodithioates. The sugar ring modification can be a modification of the 2' position of the pentose sugar. The 2'-position modification of pentose sugars includes, but is not limited to, methoxy, methoxyethoxy, propyleneoxy, and fluoro modifications (Ernst Urban, Christian R. Noe. Structural modifications of antisense oligonucleotides. II Farmaco. 2003, 58: 243-258). Base modification mainly refers to the inclusion of rare bases in single-stranded deoxynucleotides (Xiaolan Chen, Nancy Dudgeon, Long Shen and Jui H. Wang. Drug Discovery Target, 22005, 10 (8): 587-593), rare bases Refers to some bases other than A, G, C, U, including dihydrouracil (DHU), pseudouridine, and methylated 嘌呤 (mG, mA). A typical pyridinium nucleoside is linked to a C- linkage of a sugar ring by N- 1 on a heterocyclic ring, and a pseudouridine nucleoside is linked to a C-oxime of a sugar ring by C-5 on the heterocyclic ring.

One of ordinary skill in the art can modify the single-stranded deoxynucleotides of the present invention based on common general knowledge without altering its function. Such modifications include conservative additions, deletions, substitutions and modifications of the bases. Preferably, the addition, deletion, substitution and modification are carried out for 1 to 10 bases.

The addition of the single-stranded deoxynucleotide base means a single-stranded deoxynucleotide formed by adding 1 to 10 bases at one or both ends of the single-stranded deoxynucleotide provided by the present invention. The cleavage at both ends refers to a single-stranded deoxynucleotide formed by cleavage of one or several bases at one or both ends of a single-stranded deoxynucleotide provided by the present invention.

A change in a single-stranded deoxynucleotide base refers to a base change of a single-stranded deoxynucleotide provided by the present invention. A single-stranded deoxynucleotide formed.

In another aspect, the invention features a composition comprising the single-stranded deoxynucleotide for use in treating a disease caused by a viral infection.

In another aspect, the invention provides a kit for treating a disease caused by a viral infection comprising a single-stranded deoxynucleotide of the invention.

In another aspect, the invention relates to the use of said single chain deoxynucleotide for the manufacture of a medicament for the treatment of a disease caused by a viral infection.

In another aspect, the invention provides a method of treating a disease caused by a viral infection, comprising providing a therapeutically effective amount of a single chain deoxynucleotide of the invention to a subject, the effective amount being Words can be easily determined.

The application of synthetic single-stranded deoxynucleotides includes mucosal surfaces (including respiratory, digestive tract, and genitourinary mucosa) applications, subcutaneous, intramuscular injection, gastrointestinal applications, intraperitoneal applications, intravenous injections, and the like.

Viruses causing these viral infectious diseases include, but are not limited to, hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, variola virus and vaccinia virus, influenza virus, swine fever virus, hepatitis A virus, Hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and human immunodeficiency virus. Preferably, the disease caused by the viral infection is hepatitis B caused by hepatitis B virus infection and AIDS caused by human immunodeficiency virus infection.

This study demonstrates that the single-stranded deoxynucleotide stimulates the culture supernatant of human PBMC to inhibit the production of HBsAg, HBeAg and HBV nucleic acids in HepG2. 2. 15 cells. Meanwhile, the present inventors have also found that the single-stranded deoxynucleotide stimulating the culture supernatant of human PBMC can inhibit HIV-infected PBMC cells from dividing into p24 antigen. Thus, the results provided by the present invention indicate that the single-stranded deoxynucleotides inhibit viral replication and have therapeutic effects on viral infections, including but limited to HBV and HIV. In addition, it should be noted that other aspects of the present invention and inventive benefits can be directly inferred by those of ordinary skill in the art based on the disclosure of the present application. detailed description

The invention is described in further detail below in conjunction with specific examples and biological effect examples. Field It is to be understood by those of ordinary skill in the art that these examples are not intended to limit the scope of the invention. Example

In the following examples, various processes and methods not described in detail are conventional methods well known in the art, such as the synthesis using a solid phase phosphoramidite triester method.

In the following examples, the source of the reagents used, the trade name, and/or the components that are necessary to list them are indicated only once. The same reagents used hereinafter are not described above unless otherwise stated. Example 1. Synthesis of single-stranded deoxynucleotides

The synthesis of DNA fragments by, for example, solid phase phosphoramidite triester method is highly efficient and rapid, and has been widely used in DNA chemical synthesis.

DNA chemical synthesis is different from enzymatic DNA synthesis. The enzymatic DNA synthesis process extends from the 5'-3' direction, while DNA chemical synthesis starts from the 3' end. The specific reaction steps are as follows:

1) Deprotection group

The protective group dimethoxytrityl (DMT) of the nucleotide attached to the controlled-controlled porous glass (Controlled Pore Glass) was removed with Trichloroacetic Acid (TCA) to obtain a free 5'- The hydroxyl end is used for the next condensation reaction.

2) Activation

The phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protected), this intermediate will undergo a condensation reaction with the deprotected nucleotide on the controlled porous glass. ' '

3) Connection

When the phosphoramidite tetrazole active intermediate encounters a deprotected nucleotide on the controlled porous glass, it will affinity react with its 5'-hydroxyl group, condense and remove the tetrazole, and the synthesized oligonucleoside The acid chain is extended by one base forward.

4) Closed

After the condensation reaction, in order to prevent the 5'-hydroxyl group which is not involved in the reaction on the controlled porous glass from being extended in the subsequent cyclic reaction, the terminal hydroxyl group is often blocked by acetylation, and the general acetylating reagent is acetic anhydride and N. - Formed by the mixing of methyl imidazole.

5) oxidation In the condensation reaction, the nucleoside monomer is linked to the oligonucleotide attached to the controlled porous glass through the phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by acid or alkali. At this time, iodine tetrahydrofuran is commonly used. The solution converts the phosphorous amide to a phosphate triester to give a stable oligonucleotide. .

After the above five steps, a deoxynucleotide is attached to the nucleotide of the controlled porous glass, and the protective group on the newly added deoxynucleotide 5'-hydroxyl group is also removed by trichloroacetic acid. After the DMT is repeated, the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment. Finally, it is cleaved and deprotected (generally protected with benzoyl group for octa and C bases; protected with isobutyryl group for G base; T base is not protected; phosphite is protected with nitrile ethyl), purified (commonly used The HAP, PAGE, HPLC, C18, OPC and other methods, quantitative and other synthetic post-treatment can obtain oligonucleotide fragments that meet the experimental requirements.

Unvulcanized synthetic single-stranded deoxynucleotides were synthesized on an ABI 3900 DNA synthesizer; the synthesis of fully vulcanized and partially sulfurized CpG single-stranded deoxyoligonucleotides was synthesized on an ABI 394 DNA synthesizer using a displacement method.

SEQ ID NO: 1-190 was synthesized by the above method. The sequences of the single-stranded deoxynucleotides (Oligo) exemplified in the following examples are as follows:

Oligo 1 Seq No 89: 5'-TCgTCgggTgCgATCgCAgggggg-3'

01igo2 seq No 92: 5'-TCgTCgCAgAACgTTCTgggggg-3'

01igo4 seq No 95: 5'-TCgTATgCATCgATgCATAgggAgg-3'

01igo3 seq No 174: 5'-TCgTgCgACgTCgCAgATgAT-3'

01igo5 seq No 164: 5'-TCgTCggTCTTTCgAAATT-3'

01igo6 seq No 36: 5'-TCggggACgATCgTCgggggg-3'

01igo7 seq No 172: 5 '-TCgACgTTCgTCgTTCgTCgTTC-3 '

01igo8 seq No 173: 5'-TCgTCgACgTCgTTCgTTCTC-3 '

01igo9 seq No 98: 5'-TCggACgATCgTCgggggg-3'

Oligo 10 seq No 99: 5'-TCgAgCgATCgCTCgAgggggg-3'

Oligo 11 seq No 107: 5'-TCgTCgggTgCgACgTCgCAgggggg-3'

Oligo 12 seq No 170: 5'-TCgTCgggTgCgACgTCgCA-3'

Oligo 13 seq No 102: 5'-TCgTCgggTgCgACgTCgCAg-3'

Oligo 14 seq No 103: 5'-TCgTCgggTgCgACgTCgCAgg-3'

Oligo 15 seq No 104: 5'-TCgTCgggTgCgACgTCgCAggg-3' 01igol6 seq No 105: 5'-TCgTCgggTgCgACgTCgCAgggg-3'

01igol7 seq No 106: 5'-TCgTCgggTgCgACgTCgCAggggg-3'

01igol8 seq No 3: 5'-ggggTgCAACgTTCAgggggg-3'

01igol9 seq No 1 : 5'-ggggTCgTTCgTCgTTgggggg-3'

Oligo20 seq No 66: 5'-gggggCgTCgTTTTCgTCgACgAATT-3' Example 2. Single-chain deoxynucleotide stimulating human PBMC culture supernatant inhibits the secretion of HBsAg by HepG2, 2.15 cells

1. Separation of human peripheral blood mononuclear cells

1) Equipment and equipment: low temperature refrigerator, carbon dioxide incubator, ultra clean bench, inverted microscope, liquid nitrogen tank, distilled water, vacuum pump, cell culture flask, bacteria filter, filter bottle, various specifications of straw, plus Samples, droppers, blood cell counting plates, horizontal centrifuges, etc.

2) Reagents and materials: Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun. Lymphocyte stratification solution, in which sucrose-diatrizoate: specific gravity 1. 077±0, 001, purchased from Beijing Dingguo Biotechnology Co., Ltd. RPMI 1640 medium: L-glutamine RPMI1640 (GIBC0BRL) 10. g, sodium bicarbonate 2, 0 g, gentamicin 100,000 units, add three distilled water to 1000 ml, 0.22 micron filter Filtering and sterilizing and dispensing.

3) Method: Mononuclear cells of human peripheral blood were isolated using a sucrose-diatrizoate lymphocyte stratified solution. The volume ratio of stratified fluid to heparin anticoagulated peripheral blood is about 1:2. Centrifuge horizontally (1,000 X g, 20 min) o Pipette the cell layer containing mononuclear cells with a pipette and place in another centrifuge tube. Add an equal volume of serum-free medium RPMI

1640. Centrifuge at l,000 X g for 15 min and discard the supernatant. Repeat the wash twice. The supernatant was discarded, and the cells were resuspended in 2 ml of culture medium RPMI 1640, and cell count was performed.

2. Single-stranded deoxynucleotide stimulates the collection of human PBMC culture supernatant

Dilute the isolated PBMC (lymphocytes) with 10% FBS (Lanzhou Institute of Biological Products) IMM (Gibco) to a final concentration of 3 X 10 ^fi /ml, and add 2 ml, 6 X 10 ^s per well to a 12-well plate. 2ral/well; Add each single-stranded deoxynucleotide to a final concentration of 6 g/ml, and set 3 replicate wells. The 12-well plate was placed in a 37 ° C 5% CO ₂ incubator for 48 h, and the culture supernatant was collected. The culture supernatants collected from the three replicate wells were mixed together, and stored at a temperature of 20 Torr.

3. Single-chain deoxynucleotide-excited human PBMC culture supernatant inhibited the secretion of HBsAg by HepG2. 2. 15 cells (the first hospital of Jilin University)

1) Inoculate 1 X 10 ⁵ /ml HepG2. 2. 15 cells in 24-well cell culture plates, 1 ml per well, 37 °C 5 %C0 _{2 was} cultured, and at 24 h after the culture, the single-stranded deoxynucleotide-stimulated human PBMC culture supernatant was added to have a dilution factor of 1:64.

2) The culture supernatants were collected at 48h, 72h, and 96h after the culture, and the HBsAg content was determined by the hepatitis B virus e antigen diagnostic kit (Suzhou Xinbo Biotechnology Co., Ltd.). The HBsAg content was determined according to the kit. Instructions for operation.

3) Calculate the inhibition rate of HBsAg secretion: inhibition rate = (control well concentration - experimental well concentration) / control well concentration X 100%

4. Results

Single-stranded deoxynucleotide-stimulated human PBMC culture supernatants were effective in inhibiting the secretion of HBsAg by HepG2. 2.15 cells (Table 1).

5 Conclusion

Single-stranded deoxynucleotide-stimulated human PBMC culture supernatants are capable of inhibiting HBV replication, and thus the single-stranded deoxynucleotides provided by the present invention have antiviral, particularly anti-HBV biological activities. Example 3. Single-chain deoxynucleotide stimulates human PBMC culture supernatant to inhibit the secretion of HBeAg by HepG2. 2. 15 cells.

Step 1-2 is the same as step 1-2 of the embodiment 2.

3. Single-stranded deoxynucleotide-stimulated human PBMC culture supernatant inhibits the secretion of HBeAg by HepG2. 2. 15 cells.

1) Inoculate 1×10 ⁵ /ml HepG2. 2. 15 cells into a 24-well cell culture plate, lml per well, culture at 37 °C 5 % CO ₂ , and add single-stranded deoxynucleoside at 24 h after culture. The acid sputum stimulated the supernatant of PBMC to make a dilution factor of 1:64.

2) At 48h, 72h, and 96h after the culture, the culture supernatants of each group in different time periods were collected, and the HBeAg content was detected by the hepatitis B virus e antigen diagnostic kit (Suzhou Xinbo Biotechnology Co., Ltd.), HbeAg The determination of the content is carried out according to the instructions provided in the kit.

3) Calculate the inhibition rate of HBeAg secretion: inhibition rate = (control well concentration - experimental well concentration) / control well concentration X 100%

4. Results

Various single-stranded deoxynucleotide-stimulated human PBMC culture supernatants were able to effectively inhibit HepG2. 2. 15 cells secreting HBeAg at a 1:64 dilution (Table 2). 5. Conclusion: Single-stranded deoxynucleotide stimulates human PBMC culture supernatant to inhibit HBV replication, and thus the single-stranded deoxynucleotide provided by the present invention has antiviral, particularly anti-HBV biological activity. Example 4: Single-chain deoxynucleotide stimulates the inhibition of human PBMC culture supernatant on HepG2. 2. 15 cells secreting HBV DNA

Steps 1 to 2 are the same as steps 1 and 2 of Embodiment 2.

3. Single-chain deoxynucleotide-expressed human PBMC culture supernatant inhibits the secretion of HBV DNA by HepG2. 2. 15 cells.

1) Inoculate 1×10 ⁵ /ml HepG2. 2. 15 cells into a 24-well cell culture plate at 1 ml per well, and add each single-stranded deoxynucleotide at 24 h after incubation at 37 ° C 5 % CO ₂ . The human PBMC culture supernatant was stimulated to have a dilution factor of 1:64. '

2) At 48h, 72h, and 96h after culture, the culture supernatants of each group in different time periods were collected, and the HBV DNA content was detected by HBV DNA quantitative PCR kit (Shanghai Shenyou Biotechnology Co., Ltd.), HBV DNA. The determination of the content is carried out according to the instructions provided in the kit.

3) Calculate the inhibition rate of HBV DNA secretion: Inhibition rate = (control well log (copy number) - test well log (copy number) / control well log (copy number) X 100%

4. Results

Each single-stranded deoxynucleotide stimulates human PBMC culture supernatant to effectively inhibit at 1:64 dilution

HepG2. 2. 15 cells secrete HBV DNA (Table 3).

5 Conclusion

Single-stranded deoxynucleotides stimulate human PBMC culture supernatants to inhibit HBV replication, and thus the single-stranded deoxynucleotides provided by the present invention have antiviral, particularly anti-HBV, biological activities. Example 5. Anti-HIV effect of single-chain deoxynucleotides

1. Separation of human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were isolated using a sucrose-diammonia lymphocyte stratified fluid. The volume ratio of stratified fluid to heparin anticoagulated peripheral blood is about 1:2. Centrifuge horizontally (l,000 X g, 20 min). The cell layer containing hPBMC was pipetted and placed in another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at l, 000 x _g for 15 min, discard the supernatant. Repeat the wash twice. The supernatant was discarded, the cells were resuspended in 2 ml of culture medium, and cell counts were performed. 2. Culture of human peripheral blood mononuclear cells

Use 10% fetal bovine serum (Tianjin Yuyang (TBD) Biotechnology Co., Ltd.), 1% streptomycin (GIBCO), 1% glutamine (GIBCO) and 10 units of interleukin 2 per ml ( The RPMI (GIBCOBRL) of the NIH AIDS Research and Reference Reagent Program was used to culture PBMC at a cell concentration of ¹⁰ ×10 ⁶ cells/ml.

3. HIV

The HIV virus is provided by the NIH AIDS Research and Reference Reagent Program.

4. Single-chain deoxynucleotide anti-HIV experiment

96-well plate, was added at a concentration of 10X10 ⁶ cells / ml PBMC (3X 10 ⁶ cells / ml 200μ1 / well), each sample provided triplicates. After 2 days of stimulation of PBMC with single-stranded deoxynucleotides (6 g/ml), PBMCs were infected with HIV virus (0.0005 TCID50/cell), and after 6 hours of incubation (37 ° C, 5% CO ₂ incubator), cells were washed with PBS 3 After the second time, the cells were cultured, and the level of the p24 antigen in the culture supernatant was measured on the fifth day. Control wells without single-stranded deoxynucleotides were placed.

5. Determination of HIVp24 antigen level by enzyme-linked immunosorbent assay

P24 polyclonal antibody coated ELISA plate [BioMerieux BioMerieux, Human Immunodeficiency Virus Antigen Antibody (ELISA) (Aalto Bioreagents, Dublin, Eire), polyclonal antibody and p24 The antigen binds, and the p24 antigen is then combined with the added anti-p24 monoclonal antibody (provided by the kit). The anti-p24 monoclonal antibody binds to alkaline phosphatase and produces color after the addition of biphenyl diamine. The p24 antigen was obtained by cleavage of viral particles with a detergent. Specific measurement steps:

(1) Add a blocking solution to the p24 polyclonal antibody (5ng/ml in 0.1mol/L NaHCO3, ρΗ = 8.5) (2% miIkinTBS (1XTBS: 144mmol/L NaCl, 25mraol/L TRIS, _P H7.5) 300 1 / hole, sealed at room temperature for 2h;

(2) Washing the plate with PBS three times;

(3) Add 100 μl of the sample to be tested (the culture supernatant of PBMC infected) to each well of the plate. Sealed at room temperature for 2 h _;

(4) Wash the plate with PBS three times;

(5) Add 100 μl of each monoclonal antibody to the ELISA plate and add anti-ρ24 monoclonal antibody solution (provided with the kit) 0.5 g/ml in TMT I SS [2% Marvel fat free Skimmed milk powder, 20% sheep serum, 0.5% Tween 20 in TBS], sealed at room temperature for 2 h;

(6) The plate was washed three times with PBS.

(7) Add 100 μl of substrate solution (provided by the kit) to each well of the plate. TMT / SS [2% Marvel fat free skimmed milk powder, 20% sheep serum, 0.5% Tween 20 in TBS], sealed at room temperature Keep away from light.

(8) Add 50 μl of Stop Solution (provided by the kit) to each well of the plate.

(9) The absorption value of the sample at Α405 is detected by a microplate reader, and the standard curve is drawn by the measurement result and the result is calculated.

6. Results

In the culture supernatants to which different single-stranded deoxynucleotides were added, the levels of ρ24 antigen are shown in Table 4. The results showed that the concentration of ρ24 antigen secreted by the HIV virus was significantly decreased after the addition of single-stranded deoxynucleotides.

7 Conclusion

The single-stranded deoxynucleotide of the present invention is capable of inhibiting the replication of HIV virus and thereby suppressing AIDS caused by HIV infection.

1 Single-strand deoxynucleotide-stimulated human PBMC supernatant inhibited the secretion of HBsAg from HepG2.2.15 cells

48h inhibition rate 72h inhibition rate 96h inhibition rate culture solution control 0.00 0.00 0.00

Oligo 1 (1:64 dilution) 78.26 86.45 92.18

Oligo 2 (1:64 dilution) 67.98 79.54 89.78

Oligo 3 (1:64 dilution) 76.34 88.09 90, 12

Oligo 4 (1:64 dilution) 69.22 76.56 87.98

Oligo 5 (1:64 dilution) 74.59 87.06 93.03

Oligo 6 (1:64 dilution) 77.34 83.98 90.89

Oligo 7 (1:64 dilution) 65.90 73.13 88.09

Oligo 8 (1:64 dilution) 66.09 74.34 89.01

Oligo 9 (1:64 dilution) 63.89 72.41 85.67

Oligo 10 (1:64 dilution) 76.01 83.78 92, 32

Oligo 11 (1:64 dilution) 60.78 69.45 76.35

Oligo 12 (1:64 dilution) 70.32 79.46 85.78

Oligo 13 (1:64 dilution) 67.90 78.09 83.42

Oligo 14 (1:64 dilution) 70.12 78.53 89.21

Oligo 15 (1:64 dilution) 60.74 67.90 78.41

Oligo 16 (1:64 dilution) 64.09 • 69.97 • -79·.56

Oligo 17 (1:64 dilution) 69.00 78.00 85.31

Oligo 18 (1:64 dilution) 72.87 79.99 91.78

Oligo 19 (1:64 dilution) 71.78 82.34 94.07

Oligo 20 (1:64 dilution) 77.11 89.44 90.01 2 Inhibition of HepG2.2.15 cells secreting HBeAg by single-chain deoxynucleotide-stimulated human PBMC supernatant

Oligo 1 (1:64 dilution) 72.26 88.45 93.18

Oligo 2 (1:64 dilution) 66.90 80.51 90. 01

Oligo 3 (1:64 dilution) 76.12 88.21 91.12

Oligo 4 (1:64 dilution) 70.22 75.66

Oligo 5 (1:64 dilution) .73.69 86.96 94.03

Oligo 6 (1:64 dilution) 76.40

91.09

Oligo 7 (1:64 dilution) · 65.09 70.99 81.23

Oligo 8 (1:64 dilution) 70.09 80.00 89.45

Oligo 9 (1:64 dilution) 65.09 70.90 89.12

Oligo 10 (1:64 dilution) 61.90 79.34 87.96

Oligo 11 (1:64 dilution) 72.34 79.91 , 88.06

Oligo 12 (1:64 dilution) 62.09 74.45 8O O7.65

o o

Oligo 13 (1:64 dilution) 69.34 79.76 90.01

Oligo 14 (1:64 dilution) 66.11 78.54 89.00

Oligo 15 (1:64 dilution) 70.00 79.32 88.11

Oligo 16 (1:64 dilution) 63.90 76.31 86.11

Oligo 17 (1:64 dilution) 70.55 79.66 87.43

Oligo 18 (1:64 dilution) 69.38 86.99 91.21

Oligo 19 (1:64 dilution) 70.01 78.48 87.56

Oligo 20 (1:64 dilution) 67.42 79.58 90.01 Table 3 Inhibition of HBV DNA secretion by HepG2.2.15 cells by single-chain deoxynucleotide-stimulated human PBMC supernatant

Oligo 1 (1:64 dilution) 67.24 79.14 88.09

Oligo 2 (1:64 dilution) 69.18 80.01 92.06

Oligo 3 (1:64 dilution) 71.11 86.32 91.37

Oligo 4 (1:64 dilution) 72.89 85. '32 90.97

Oligo 5 (1:64 dilution) 70.65 84.36 90.06

Oligo 6 (1:64 dilution) 69.78 78.35 89.76

Oligo 7 (1:64 dilution) 66.09 71.99 82.13

Oligo 8 (1:64 dilution) 71.19 81.00 89.75

Oligo 9 (1:64 dilution) 64.09 71.02 84.12

Oligo 10 (1:64 dilution) 64.90 79.44 88.96

Oligo 11 (1:64 dilution) 70.24 79.44 86.36

Oligo 12 (1:64 dilution) 63.19 74.55 86.69

Oligo 13 (1:64 dilution) 70.24 81.12 90.01

Oligo 14 (1:64 dilution) 67.12 78.59 89.21

Oligo 15 (1:64 dilution) 71.00 79.42 88.71

Oligo 16 (1:64 dilution) 64.32 76.31 85.17

Oligo 17 (1:64 dilution) 71.55 79.86 87.88

Oligo 18 (1:64 dilution) 68.38 86.99 90.21

Oligo 19 (1:64 dilution) 71.01 78.48 87.56

Oligo 20 (1:64 dilution) 65.55 79.21 91.01 Table 4 Levels of p24 antigen production when different single-stranded deoxynucleotides were added

Oligos p24 antigen (ng/ml)

Oligo-1 13

Oligo- 2 9

Oligo - 3 17

Oligo- 4 11

Oligo- 5 6

Oligo - 6 9

01igo-7 16

01igo-8 5

Oligo- 9 7

Oligo-10 8

Oligo - 11 18

Oligo- 12 10

Oligo- 13 4

Oligo-14 8

Oligo- 15 6

Oligo- 16 9

Oligo- 17 10

Oligo-18 11

Oligo-19 6

Oligo-20 9

Control 32

Claims

Rights request

What is claimed is: 1. A synthetic single-stranded deoxynucleotide having a sequence represented by one of the following formulas (i) to (v), or a sequence of one of the following (i) to (v) A sequence in which a 10 base is conservatively substituted, added, deleted, or modified:

(i) (G)n(L)n X1X2CGY1 Y2(M)n (G)n, where X1=A,T,G; X2= A,T _; Y1= A,T; Y2= A,T,C ; L, M = A, T, C, G; n is 0-6;

(ii) (G)n(L)nCG(XY)nCG(M)n(G)n, where X=A,T; Y= A,T _; L,M=A,T,C,G; n for

0-6;

(iii) (TCG)n(L)nCG (M)n(G)n, where L, M=A, T, C, G; n is 0-6;

(iv) (TCG)n(L)nXlX2CG(M)n, where X1=A,T,G; X2= A,T; L,M=A,T,C,G; n is

0-6;

(v) A sequence containing TTCGTCG.

2. The single-stranded deoxynucleotide of claim 1 having the sequence set forth in one of SEQ ID NO. 1-190. 3. The single-stranded deoxynucleotide of claim 2 having SEQ ID NO: 1,

3, 36, 66, 89, 92, 95, 98,

The sequence shown in one of 99, 102, 103, 104, 105, 106, 107, 164, 170, 172, 173, 174.

The single-stranded deoxynucleotide according to any one of claims 1 to 4, which is chemically modified, which is selected from the group consisting of a phosphate skeleton modification, a sugar ring modification, and a base modification.

5. The single-stranded deoxynucleotide of claim 4, wherein said phosphate backbone modification is a modification of a thio- or phosphate linkage, said saccharide modification being a modification of the 2' position of the pentose.

6. The single-stranded deoxynucleotide of claim 5, wherein the modification of the phosphate bond is a modification of a methyl phosphate, a selenophosphate, a boronyl phosphate or a phosphorodithioate.

7. The single-stranded deoxynucleotide of claim 5, wherein the 2'-position modification of the pentose sugar is a modification of a methoxy group, a methoxyethoxy group, a propyleneoxy group or a fluoro modification.

8. The single-stranded deoxynucleotide of claim 1, wherein the modification of the base is a rare base comprising dihydrouracil, pseudouracil, and methylated purine.

9. A composition comprising the single-stranded deoxynucleotide of any one of claims 1-8.

A kit for treating a disease caused by a viral infection, which comprises the single-chain deoxynucleotide of any one of claims 1-8.

11. The kit of claim 10, wherein the virus is selected from the group consisting of hepatitis B virus, TTV virus, adenovirus, Papillomavirus, herpes zoster virus, variola virus and vaccinia virus, influenza virus, swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola, enterovirus and human immunodeficiency virus.

The use of the single-chain deoxynucleotide according to any one of claims 1 to 8 for the preparation of a medicament for treating a disease caused by a viral infection.

13. The use of claim 12, wherein the virus is selected from the group consisting of hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, variola virus and vaccinia virus, influenza virus, swine fever virus, hepatitis A Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Hepatitis G Virus, Rabies Virus, Ebola Virus, Enterovirus and Human Immunodeficiency Virus.

A method of treating a disease caused by a viral infection, comprising providing a subject with a therapeutically effective amount of the single-chain deoxynucleotide of any one of claims 1-8.

15. The method of claim 12, wherein said single-stranded deoxynucleotide is applied to a mucosal surface, subcutaneously, intramuscularly, gastrointestinally, intraperitoneally or intravenously.

16. The method of claim 14 or 15, wherein the virus is selected from the group consisting of hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, variola virus and vaccinia virus, influenza virus, swine fever virus, nail Hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus and human immunodeficiency virus.