Original Article Silencing and DNA

P. Parveen*, K. Deepti Brundavani, K. Mahathi, M.S. Bhavani and SK. Shaheda Sultana

Hindu College of Pharmacy, Amaravathi Road, Guntur-522002, Andhra Pradesh, India

ABSTRACT

This review revealed a new mechanism for gene regulation through “” at the transcriptional level (TGS) or at the post- transcriptional level (PTGS), which play a key role in many essential cellular processes. Today dsRNA is used as a powerful tool to experimentally elucidate the function of essentially any gene in a cell. The immense impact of the discovery of RNA interference (RNAi) on biomedical research and its novel medical applications in the future are reviewed in this article, with particular stress on therapeutic applications of radio-labeled antisense (RASONs) for diagnosis and treatment of various and neurodegenerative diseases by “gene silencing”. Antisense oligonucleotides (ASONs) can also modulate

which 74% of all human undergo. Epigenetic changes affect Address for chromatin structure and thus regulate processes such as , Correspondence X- inactivation, allele-specific expression of imprinted genes, and inactivation of tumor suppressor genes. Treatment with Hindu College of inhibitors of DNA methylation and deacetylation can Pharmacy, Amaravathi reactivate epigenetically silenced genes and has been shown to Road, Guntur-522002, restore normal gene function. In cells, this results in Andhra Pradesh, India. expression of tumor suppressor genes and other regulatory functions, E-mail: inducing growth arrest and apoptosis. pothukantiparveen Keywords: RNAi, X-chromosome RASONs, DNA methylation, @gmail.com Gene silencing.

INTRODUCTION machinery1 in the cell. Genes are regulated Gene silencing is a general term at either the transcriptional or post- describing epigenetic processes of gene transcriptional level. (See fig. 1.) regulation. The term gene silencing is Transcriptional gene silencing is the generally used to describe the "switching result of histone modifications, creating an off" of a gene by a mechanism other than environment of heterochromatin26 around a genetic24 modification. That is, a gene which gene that makes it inaccessible to would be expressed (turned on) under transcriptional machinery (RNA polymerase, normal circumstances is switched off by transcription factors, etc.).

American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org Parveen et al______ISSN 2321 – 2748

Post-transcriptional gene silencing is introduced into cells can induce suppression the result of mRNA of a particular gene being of specific genes of interest. RNAi may also destroyed. The destruction of the mRNA be used for large-scale screens that prevents to form an active gene systematically shut down each gene in the product (in most cases, a ). A common cell, which can help identify the components mechanism of post-transcriptional28 gene necessary for a particular cellular process7 or silencing is RNAi. an event such as cell division. Exploitation of Both transcriptional and post- the pathway is also a promising tool in transcriptional gene silencing are used to biotechnology6 and medicine. regulate endogenous genes. Mechanisms of gene silencing also protect3 the organism's DNA methylation from transposons and . Gene DNA methylation is a biochemical silencing thus may be part of an ancient process that is important for normal immune system protecting from such development in higher organisms. It involves infectious DNA elements. the addition of through cell a methyl group purine rin to the 5 position of the RNA interference ring or the number 6 nitrogen of The enzyme trims double the adenineg (cytosine and are two of stranded RNA, to form small interfering RNA the four bases of DNA). This modification or micro RNA. These processed are can be inherited division. incorporated into the RNA-induced silencing DNA methylation is a crucial part of complex (RISC), which targets messenger normal organismal development2 and cellular RNA to prevent translation. differentiation in higher organisms. DNA RNA interference (RNAi) is a methylation stably alters the mechanism that inhibits gene expression by pattern in cells such that cells can "remember causing the degradation of specific RNA where they have been" or decrease gene molecules or hindering the transcription of expression; for example, cells programmed to specific genes. The targets are often RNA be pancreatic islets during embryonic from viruses and transposons14 (probably a development remain pancreatic islets form of innate immune response), although it throughout the life of the organism without also plays a role in regulating16 development continuing signals telling them that they need and genome maintenance. Key to the RNAi to remain islets. DNA methylation is typically processes are small interfering RNA strands removed during formation and re- (siRNA), which have complementary established through successive cell divisions sequences to a targeted RNA during development. However, the latest strand. The siRNA "guides" proteins5 within research shows that hydroxylation of methyl the RNAi pathway to the targeted messenger group occurs rather than complete removal of RNA (mRNA) and "cleaves" them, breaking methyl groups in zygote. Some methylation them down into smaller portions that can no modifications that regulate gene expression longer be translated24 into protein. A type of are inheritable and are referred to as RNA transcribed from the genome itself, epigenetic regulation. miRNA, and works in the same way. In addition, DNA methylation The selective and robust effect of suppresses the expression of viral genes42 and RNAi on gene expression makes it a valuable other deleterious elements that have been research tool, both in and in living incorporated into the genome of the host over organisms because synthetic dsRNA time. DNA methylation also forms the basis

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748 of chromatin structure, which enables cells to transcriptional state of the gene. Exceptions to form the myriad characteristics necessary for this rule, however, can be found in multi cellular life from a single immutable mammalian cells where these regions are sequence of DNA. Methylation also plays a methylated to maintain transcriptional crucial role in the development of nearly all inactivation. Thus, CpG islands in promoters types of cancer.DNA methylation at the 5 of genes located on the inactivated X position of cytosine has the specific effacing chromosome of females are methylated, as gene expression and has been found in every are certain imprinted genes in which only the vertebrate examined. In adult ct of redu maternal or paternal allele is expressed. somatic tissues, DNA methylation typically Epigenetic effects such as occurs in a CpG dinucleotide context; non- hypermethylation can also induce inevitable CpG methylation9 is prevalent in embryonic alterations in gene xpression. Methylation of stem cells. the DNA repair genes MLH1 and MGMT can lead to their inactivation, methylation may Mechanism of DNA methylation affect the transcription of genes in two ways. In mammalian DNA, 5-methylcyto- First, the methylation of DNA itself may sine is found in approximately 4% of genomic physically impede the binding of DNA, primarily at cytosine-guanosine transcriptional to the gene, and dinucleotides (CpGs). Such CpG sites occur second, and likely more important, at lower than expected frequencies throughout methylated DNA may be bound by proteins the but are found more known as methyl-CpG-binding domain frequently at small stretches of DNA called proteins (MBDs). MBD proteins then recruit CpG islands. These islands are typically additional proteins to the locus, such as found in or near regions of genes, histone deacetylases and other chromatin where transcription is initiated. In contrast to remodeling proteins that can modify , the bulk of genomic DNA, in which most thereby forming compact, inactive chromatin, CpG sites are heavily methylated, CpG termed . This link between islands in germ-line tissue and promoters of DNA methylation and chromatin structure is normal somatic cells remain un-methylated, very important. In particular, loss of methyl- allowing gene expression to occur. (See fig. CpG-binding protein 2 (MeCP2) has been 2.) implicated in ; and DNA methylation helps to maintain medimethyl-CpG-binding domain protein 2 transcriptional silence in non expressed or (MBD2) ates the transcriptional silencing of non coding regions of the genome. For hypermethylated genes in cancer. Research example, pericentromeric11 heterochromatin, has suggested that long-term storage which is condensed and transcriptionally in humans may be regulated by DNA inactive, is heavily methylated. Hyper methylation. methylation thus ensures this DNA is late- replicating and transcriptionally quiescent, Regulation of DNA methylation and suppresses the expression of any DNA methylation is controlled at potentially harmful viral sequences or several different levels in normal and tumor transposons that may have integrated into cells. The addition of methyl groups is carried such sites containing highly repetitive out by a family of enzymes, DNA sequences. By contrast, these sites are (DNMTs). Chromatin generally un-methylated in promoter regions structure in the vicinity of gene promoters of euchromatin, regardless of the also affects DNA methylation and

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748 transcriptional activity. These are in turn Histone deacetylation regulated by various factors such as Although methylation controls gene spacing and histone acetylases13, activity, it alone is insufficient to repress which affect access to transcriptional factors. transcription. The local chromatin structure also contributes in determining whether genes DNA methyltransferases are transcribed or repressed. For example, In mammalian cells, DNA DNMTs may regulate gene silencing methylation occurs mainly at the C5 position accompanying promoter DNA methylation by of CpG dinucleotides and is carried out by of recruiting histone deacetylases (HDACs) and enzymatic activities–maintenance methylation other chromatin-binding proteins to promoter and de novo methylation. Two general sites to maintain histone deacetylation. classes. The state of histone acetylation is Maintenance methylation activity is important in regulating chromatin17 structure necessary to preserve DNA methylation after and gene transcription. The chromatin every cellular DNA replication cycle. Without structure surrounding un-methylated, the DNA (DNMT), the transcriptionally active genes differs from that replication machinery itself would produce of methylated, silenced genes. Both daughter strands that are un-methylated and, nucleosome structure and histone acetylation over time, would lead to passive affect chromatin structure and thus regulate demethylation. DNMT1 is the proposed gene transcription. are maintenance methyltransferase that is composed of 146 base pairs of DNA wound responsible for copying DNA methylation15 around complexes of eight histones within the patterns to the daughter strands during DNA nucleus. In CpG islands that are un- replication. Mouse models with both copies methylated, the nucleosomes are in a more of DNMT1 deleted are embryonic lethal at open configuration that allows access of approximately day 9, due to the requirement factors that favor transcription When CpG of DNMT1 activity for development in islands are hypermethylated, as in mammalian cells. heterochromatin, the nucleosomes become It is thought that DNMT3a and more tightly compacted, inhibiting the access DNMT3b are the de novo methyltransferases of regulatory proteins that promote that set up DNA methylation patterns early in transcription. Interestingly, recent studies development. DNMT3L is a protein that is have determined that methylation of specific homologous to the other DNMT3s but has no residues on the tails of histone H3 are critical catalytic activity. Instead, DNMT3L assists for maintaining the appropriate chromatin the de novo methyltransferases by increasing configuration. Methylation of lysine 9 their ability to bind to DNA and stimulating residues on H3 facilitates transcriptional their activity. Finally, DNMT2 (TRDMT1) repression, whereas methylation of lysine 4 has been identified as a DNA methyl- on H3 is associated with transcriptionally transferase homolog, containing all 10 active euchromatin. (See fig. 3.) sequence motifs common to all DNA methyltransferases; however, DNMT2 Histone deacetylation inhibitors (TRDMT1) does not methylate DNA but Given that HDACs are integral to instead methylates cytosine-38 in the maintaining transcriptional silence, they can anticodon loop of aspartic acid transfer RNA. increase gene expression in the absence of hypermethylation19. A variety of HDAC inhibitors have been shown to inhibit histone

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748 deacetylation in human tumor cells, leading to mutations are more commonly observed. accumulation of acetylated histone proteins. Subsequent loss of the remaining allele In leukemic cells, treatment with HDAC through deletion, , or loss of inhibitors results in growth arrest, heterozygosity can eliminate the remaining differentiation, or apoptosis. These drugs hold functional gene. Inactivation of regulatory promise for cancer therapy, especially when genes in this manner can lead to the used in combination with methylation development of cancer. inhibitors, other differentiating agents, or DNA methylation is an important cytotoxic compounds. HDAC inhibitors regulator of gene transcription and a large undergoing clinical evaluation for treatment body of evidence has demonstrated that of hematopoietic malignancies include the aberrant DNA methylation is associated with natural HDAC inhibitor trichostatin, unscheduled gene silencing, and the genes hydroxamic acid derivatives (such as with high levels of 5-methylcytosine23 in their suberanilohydroxamic acid), the cyclic promoter region are transcriptionally silent. tetrapeptide depsipeptide50, benzamide DNA methylation49 is essential during derivatives like MS-275 and CI-994, and , and in somatic cells, aliphatic acids such as valproic acid and patterns of DNA methylation are generally phenylbutyrate. transmitted to daughter cells with a high It should be noted that methylation is fidelity. Aberrant DNA methylation patterns dominant to histone deacetylation, so have been associated with a large number of transcription cannot occur without first human malignancies and found in two distinct inhibiting methylation. Initial treatment with forms: hypermethylation and hypomethyla- or decitabine, followed by HDAC tion compared to normal tissue. inhibitors, can produce additive or synergistic Hypermethylation is one of the major effects for re-expression of transcriptionally epigenetic25 modifications that repress silenced genes These preclinical findings, transcription via promoter region of tumour coupled21 with the above model of chromatin suppressor genes. Hypermethylation typically structure and its regulation by both occurs at CpG islands in the promoter region methylation and acetylation, provide the and is associated with gene inactivation. rationale for therapeutic approaches in which Global hypomethylation has also been the two processes are simultaneously implicated in the development and targeted22. progression27 of cancer through different mechanisms. (See fig. 4.) Gene methylation in cancer According to the widely accepted Demethylation and gene reactivation 'two-hit' hypothesis of The demethylating agent azacitidine proposed by Knudson, loss of function of and its deoxy derivative, decitabine, are both alleles in a given gene (e.g. a tumor powerful inhibitors of DNA methylation. suppressor) is required for malignant Preclinical studies have shown that after transformation. The first hit typically is in the cellular uptake and , form of a mutation in a critical gene, followed azacitidine is incorporated into RNA, where it by loss of the wild-type allele through suppresses RNA synthesis and has cytotoxic deletion32 or loss of heterozygosity. In effects. After conversion to 5-aza-2'- familial cancers this first hit may occur diphosphate47 by ribonucle- through germ-line mutations, while in otide reductase and subsequent noninherited sporadic cancers somatic phosphorylation, the triphosphate form is

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748 incorporated into DNA in place of the natural DNA methylation. It is based on base cytosine. Because of the substitution of aforementioned sodium bisulfite the 5' nitrogen atom in place of the carbon, conversion of genomic DNA, which is the DNMTs are trapped29 on the substituted then sequencing on a Next-generation DNA strand and methylation is inhibited. sequencing platform. The sequences Thus, in the presence of these analogs, a obtained are then re-aligned to the significant proportion of the DNA becomes reference genome to determine hemimethylated41. A second round of DNA methylation states of CpG dinucleotides synthesis in the presence of these drugs based on mismatches resulting from the results in full double-stranded DNA conversion of unmethylated into demethylation while both azacitidine and . decitabine inhibit methylation, they do differ  The HELP assay, which is based on in some respects. Because it contains a ribose restriction enzymes' differential ability to moiety, azacitidine can be phosphorylated by recognize and cleave methylated and uridine-cytidine kinase, and when this unmethylated CpG DNA sites. happens it is subsequently incorporated into  Now rarely-used assay based upon RNA. Since decitabine contains a restriction enzymes' differential recogni- 31 deoxyribose group, it is incorporated only tion of methylated and unmet ChIP-on- into DNA. While some have suggested that chip assays, which is based on the ability differential incorporation into RNA and DNA of commercially prepared antibodies to may account for differences in methylation bind to DNA methylation-associated and toxicity profiles between these two proteins33 like MeCP2. agents, there are no direct clinical data at  Restriction landmark genomic scanning, a present to support this proposition. complicated and ylated CpG sites; the assay is similar in concept to the HELP Inhibition of Methylation with azacytidine assay. See figure 5.  Methylated DNA (MeDIP), analogous to chromatin Detectors immunoprecipitation, immunoprecipi- DNA methylation can be detected by tation is used isolate methylated DNA the following assays currently used in fragments for input into DNA detection scientific research: methods such as to DNA microarrays  Methylation-Specific PCR (MSP), which (MeDIP-chip) or DNA sequencing is based on a chemical reaction of sodium (MeDIP-seq). 39 bisulfite with DNA that converts un- methylated cytosines of CpG Future challenges dinucleotides to uracil or UpG, followed  The age in which becomes an by traditional PCR. However, methylated essential component in the clinical cytosines will not be converted in this management of the clinical management process, and primers are designed to of the oncology patient is getting closer overlap the CpG site of interest, which  Treatment of age macular degeneration allows one to determine methylation  Prevents damage caused by status as methylated or un-methylated. huntingtons disease  Whole genome , also  Treatment of alzimers disease and spinal known as BS-Seq, which is a high- cord injury throughput genome-wide analysis of

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

 Treatment of developmental abnormali- 4. Mello CC, C onte D. Revealing the world of ties. RNA interference 2004; 431:338- 342. CONCLUSION 5. Meuster G, T uschl T . Mechanisms of gene silencing by double stranded RNA. Nature

2004; 431:343-9. The biological significance of DNA 6. Hammond SM. D icing and Slicing. T he methylation in the regulation of gene core machinery of the RNA inter ference expression and its role in cancer is pathway. FE BS Letters 2005; 579: 5822-9. increasingly recognized. It is now clear that 7. Plastre K. RHA RNA silencing: the these epigenetic changes affect chromatin Genome’s immune system. Science 2002; structure and thus regulate processes such as 296:1263-65. transcription, X-chromosome inactivation, 8. Dorsett Y, T uschl T. SiRNA- applications in allele-specific expression of imprinted genes, functional and potentials in and inactivation of tumor suppressor genes. therapeutic. Nature Reviews 2004. Treatment with inhibitors of DNA 9. Hannon GJ, Rose JJ. Unlocking the potential of the human genome with RNA methylation and histone deacetylation can interference. Nature 2004; 431:371-8. reactivate epigenetically silenced genes and 10. Soutschek J, et al. T herapeutic silencing of has been shown to restore normal gene an endogenomic gene by systemic function. In cancer cells, this results in administration of modified SiRNA. Nature expression of tumor suppressor genes and 2004, 432, 173-178. other regulatory functions, inducing growth 11. Morrsey D V, et al. Potent and persistent arrest and apoptosis. Azacitidine and invivo antiHBV activity of chemically decitabine are clinically active in MDS and modified siRNA. Nature 2005;23: 1002-7. other hematologic malignancies and may 12. Simmerman T S, et al. RNAi mediated gene have therapeutic potential in other malignant silencing in nonhuman primates. Nature disorders. Continued clinical trials with 2006; 441:111-4. hypomethylating agents and HDAC 13. Deewanjee Mrinal K, et al. D evelopment of sensitive radioiodinated antisense inhibitors, alone or in combination, may probes by conjugation provide further advances in the treatment of techniques. Bioconjug C hem 1992; 2:195- hematopoietic malignancies and solid tumors. 200. Epigenetics is everywhere you can 14. Hnatowich D J. Antisense imaging. Where inherit something beyond DNA sequence are we now? Cancer Biother Radiopharm that’s were the real excitement in genetics 2000; 15:447-57. now. 15. Askari F K, Mc D onnell WM. Antisense oligonuclotide therapy. NEJM 1996; REFERENCES 334:316-8. 16. Wickstrom E. C linical trials of genetic therapy with antisense D NA and D NA 1. Bertil D aneholt. T he Nobel Prize in vectors. Ibid- ASON for cMYC mRNA in physiology and Medicine 2006. The Nobel Assembly at Karolinska Institute. Burkitt’s lymphoma. 17. Nature clinical practice oncology 2. Fire A, XuS, Montgomery MK, Kostas SA, methylation and gene silencing in cancer. D river SE and Mello- CC: Potent and specific genetic inter ference by double 18. The epigenetic progenitor origin of human cancer. Andrew P. Feinberg Nature Reviews, stranded RNA in aenorhabditis elegans. volume. Nature 1998; 391:806-11. 19. Epigenetics and cancer treatment. european 3. Hannon GL. RNA interference. Nature journal of pharmacology. 2002; 418:244-51.

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

20. The cotext and potential of epigenetics in 30. Li, J.; Pei, H.; Zhu, B.; Liang, L.; Wei, M.; oncology. J Lopez, M perchade, HM coley , He, Y.; Chen, N.; Li, D.; Huang, Q.; Fan, C. A Webb and T crook. Self-assembled multivalent DNA 21. McLaughlin, C. K.; Hamblin, G. D.; nanostructures for noninvasive intracellular Sleiman, H. F. Supramolecular DNA delivery of immunostimulatory CpG assembly Chem. Soc. Rev. 2011, 40, 5647– oligonucleotides ACS Nano 2011, 5, 8783– 56. 8789. 22. Fu, J.; Liu, M.; Liu, Y.; Yan, H. Spatially- 31. Jiang, Q.; Song, C.; Nangreave, J.; Liu, X.; interactive biomolecular networks organized Lin, L.; Qiu, D.; Wang, Z.-G.; Zou, G.; by nanostructures Acc. Chem. Liang, X.; Yan, H.; Ding, B.DNA origami as Res. 2012, 45, 1215–26. a carrier for circumvention of drug 23. Campolongo, M. J.; Tan, S. J.; Xu, J.; resistance J. Am. Chem. Soc. 2012, 134, Luo, D.DNA nanomedicine: Engineering 13396–13403. DNA as a polymer for therapeutic and 32. Kim, K. R.; Kim, D. R.; Lee, T.; Yhee, J. Y.; diagnostic applications Adv. Drug Delivery Kim, B. S.; Kwon, I. C.; Ahn, D. R. Drug Rev. 2010, 62,606–616. delivery by a self-assembled DNA 24. Smith, D.; Schuller, V.; Engst, C.; Radler, J.; tetrahedron for overcoming drug resistance Liedl, T. Nucleic acid nanostructures for in breast cancer cells Chem Comm. 2013, biomedical applications Nanomedi- 49, 2010–2. cine. 2013, 8, 105-21. 33. Modi, S.; M, G. S.; Goswami, D.; Gupta, G. 25. Hamblin, G. D.; Carneiro, K. M.; D.; Mayor, S.; Krishnan, Y.A DNA Fakhoury, J. F.; Bujold, K. E.; Sleiman, H. F. nanomachine that maps spatial and temporal Rolling circle amplification-templated DNA pH changes inside living cells Nat. nanotubes show increased stability and cell Nanotechnol. 2009, 4,325–30. penetration ability J. Am. Chem. 34. Conway, J. W.; McLaughlin, C. K.; Soc. 2012, 134, 2888–91. Castor, K. J.; Sleiman, H.DNA nanostructure 26. Walsh, A. S.; Yin, H.; Erben, C. M.; serum stability: Greater than the sum of its Wood, M. J. A.; Turberfield, A. J.DNA cage parts Chem. Commun. 2013, 49, 1172–4. delivery to mammalian cells ACS 35. Edwardson, T. G. W.; Carneiro, K. M. M.; Nano 2011, 5, 5427–5432. McLaughlin, C. K.; Serpell, C. J.; 27. Lee, H.; Lytton-Jean, A. K. R.; Chen, Y.; Sleiman, H. F. Site-specific positioning of Love, K. T.; Park, A. I.; Karagiannis, E. D.; dendritic alkyl chains on DNA cages enables Sehgal,A.; Querbes, W.; Zurenko, C. S.; their geometry-dependent self-assembly Nat. Jayaraman, M.; Peng, C. G.; Charisse, K.; Chem. 2013, 5, 868–875. Borodovsky, A.; Manoharan, M.; 36. Lo, P. K.; Karam, P.; Aldaye, F. A.; Donahoe, J. S.; Truelove, J.; Nahrendorf, M.; McLaughlin, C. K.; Hamblin, G. D.; Langer, R.; Anderson, D. G. Molecularly Cosa, G.; Sleiman, H. F. Loading and self-assembled nucleic acid nanoparticles for selective release of cargo in DNA nanotubes targeted in vivo siRNA delivery Nat. with longitudinal variation Nat. Chem. 2010, Nanotechnol. 2012, 7, 389–393. 2, 319–28. 28. Keum, J.-W.; Ahn, J.-H.; Bermudez, H. 37. Keum, J.-W.; Bermudez, H. Enhanced Design, assembly, and activity of antisense resistance of DNA nanostructures to DNA nanostructures Small 2011, 7, 3529– enzymatic digestion Chem. Commun. 3535. 2009, 7036–7038. 29. Schuller, V. J.; Heidegger, S.; Sandholzer, 38. Davidson, B. L.; McCray, P. B., Jr.Current N.; Nickels, P. C.; Suhartha, N. A.; prospects for RNA interference-based Endres, S.; Bourquin, C.; Liedl, T. Cellular therapies Nat. Rev.: Genet. 2011, 12, 329– immunostimulation by CpG-sequence- 40. coated DNA origami structures ACS 39. DeVos, S. L.; Miller, T. M. Antisense Nano 2011, 5, 9696–9702. oligonucleotides: Treating neuro-

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

degeneration at the level of of antisense oligonucleotides by delivery as RNA Neurotherapeutics 2013, 10, 486–97. double stranded complexes Biochem. 40. Deleavey, G. F.; Watts, J. K.; Alain, T.; Pharmacol. 2004, 68(3) 403–7. Robert, F.; Kalota, A.; Aishwarya, V.; 45. Bielinska, A.; Shivdasani, R. A.; Zhang, L. Pelletier, J.; Gewirtz, A. M.; Sonenberg, N.; Q.; Nabel, G. J. Regulation of gene Damha, M. J. Synergistic effects between expression with double-stranded analogs of DNA and RNA improve the phosphorothioate oligonucleotides potency of siRNA-mediated gene Science 1990, 250, 997–1000. silencing Nucleic Acids Res. 2010, 38, 4547– 46. Kim, S. H.; Jeong, J. H.; Lee, S. H.; Kim, S. 4557. W.; Park, T. G.PEG conjugated VEGF 41. Oliveira, C. L.; Juul, S.; Jorgensen, H. L.; siRNA for anti-angiogenic gene therapy J. Knudsen, B.; Tordrup, D.; Oteri, F.; Controlled Release 2006, 116, 123–9. Falconi, M.; Koch, J.; Desideri, A.; 47. Breunig, M.; Hozsa, C.; Lungwitz, U.; Pedersen, J. S.; Andersen, F. F.; Knudsen, B. Watanabe, K.; Umeda, I.; Kato, H.; R. Structure of nanoscale truncated Goepferich, A. Mechanistic investigation of octahedral DNA cages: Variation of single- poly (ethylene imine)-based siRNA delivery: stranded linker regions and influence on Disulfide bonds boost intracellular release of assembly yields ACS Nano 2010, 4, 1367– the cargo J. Controlled Release 2008, 76. 130, 57–63. 42. Ferrari, N.; Bergeron, D.; Tedeschi, A. L.; 48. Josse J, Kaiser Ad, kornberg A. Enzymatic Mangos, M. M.; Paquet, L.; Renzi, P. M.; synthesis of deoxyribonucleic acid. VIII. Damha, M. J. Characterization of antisense Frequencies of nearest neighbor base oligonucleotides comprising 2′-deoxy-2′- sequences in deoxyribonucleic acid. J Biol fluoro-beta-d-arabinonucleic acid (FANA): Chem. 1961 Mar; 236:864–875. [PubMed] Specificity, potency, and duration of 49. Swartz Mn, Trautner Ta, Kornberg A. activity Ann. N.Y. Acad. Sci. 2006, 1082, 91– Enzymatic synthesis of deoxyribonucleic 102. acid. XI. Further studies on nearest neighbor 43. Kanaori, K.; Tamura, Y.; Wada, T.; base sequences in deoxyribonucleic acids. J Nishi, M.; Kanehara, H.; Morii, T.; Biol Chem. 1962 Jun; 237:1961– Tajima, K.; Makino, K. Structure and 1967. [PubMed]. stability of the consecutive stereoregulated 50. Bird AP, Taggart MH. Variable patterns of chiral phosphorothioate DNA total DNA and rDNA methylation in duplex Biochemistry 1999, 38, 160. animals. Nucleic Acids Res. 1980 Apr 11; 44. Astriab-Fisher, A.; Fisher, M. H.; 8(7):1485–1497. [PMC free article] Juliano, R.; Herdewijn, P. Increased uptake [PubMed].

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

Figure 1. Transcriptional RNA polymerase

Figure 2. HDC attaches via meCP

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

Figure 3. The mechanism whereby DNA methylation and histone deacetylation cooperate to repress transcription

AJPCT[2][5][2014]544-555 Parveen et al______ISSN 2321 – 2748

Figure 4. Altered DNA-methylation patterns in tumorigenesis

Figure 5. Pathway for the methylation of cytosine in the mammalian genome and effect of inhibiting methylation with 5- azacytidine

AJPCT[2][5][2014]544-555