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Molecular and Cellular Biochemistry

, Volume 427, Issue 1–2, pp 157–167 | Cite as

Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells

  • Akhil Varshney
  • Jyoti Bala
  • Baby Santosh
  • Ashima Bhaskar
  • Suresh Kumar
  • Pramod K. Yadava
Article

Abstract

Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

Keywords

Telomerase TRAP assay SELEX Cancer RNA aptamer Therapeutics 

Abbreviations

hTR

Human telomerase RNA component

hTERT

Human telomerase reverse transcriptase

SELEX

Systematic evolution of ligand by exponential enrichment

Notes

Acknowledgements

The present study was supported by Project Grants from Department of Sciences and Technology and Grant received under UGC Resource Networking Centre, Government of India.

References

  1. 1.
    Greider CW, Blackburn EH (1987) The telomere terminal transferase of tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51:887–898. doi: 10.1016/0092-8674(87)90576-9 CrossRefPubMedGoogle Scholar
  2. 2.
    Blackburn EH (2005) Telomeres and telomerase: Their mechanisms of action and the effects of altering their functions. FEBS Lett 579(4):859–862CrossRefPubMedGoogle Scholar
  3. 3.
    Autexier C, Lue NF (2006) The structure and function of telomerase reverse transcriptase. Annu Rev Biochem 75:493–517. doi: 10.1146/annurev.biochem.75.103004.142412 CrossRefPubMedGoogle Scholar
  4. 4.
    Nakamura TM, Morin GB, Chapman KB et al (1997) Telomerase catalytic subunit homologs from fission yeast and human. Science 277:955–959. doi: 10.1126/science.277.5328.955 CrossRefPubMedGoogle Scholar
  5. 5.
    Sharma GG, Gupta A, Wang H et al (2003) hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 22:131–146. doi: 10.1038/sj.onc.1206063 CrossRefPubMedGoogle Scholar
  6. 6.
    Poole JC, Andrews LG, Tollefsbol TO (2001) Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene 269:1–12. doi: 10.1016/S0378-1119(01)00440-1 CrossRefPubMedGoogle Scholar
  7. 7.
    Autexier C, Bachand F (2001) Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions. Mol Cell Biol 21:1888–1897. doi: 10.1128/MCB.21.5.1888-1897.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lai CK, Mitchell JR, Collins K (2001) RNA binding domain of telomerase reverse transcriptase. Mol Cell Biol 21:990–1000. doi: 10.1128/MCB.21.4.990-1000.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Moriarty TJ, Huard S, Dupuis S, Autexier C (2002) Functional multimerization of human telomerase requires an RNA interaction domain in the N terminus of the catalytic subunit. Mol Cell Biol 22:1253–1265. doi: 10.1128/MCB.22.4.1253 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Moriarty TJ, Ward RJ, Taboski MAS, Autexier C (2005) An anchor site-type defect in human telomerase that disrupts telomere length maintenance and cellular immortalization. Mol Biol Cell 16:3152–3161. doi: 10.1091/mbc.E05-02-0148 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bryan TM, Goodrich KJ, Cech TR (2000) Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase. Mol Cell 6:493–499. doi: 10.1016/S1097-2765(00)00048-4 CrossRefPubMedGoogle Scholar
  12. 12.
    Banik SSR, Guo C, Smith AC et al (2002) C-terminal regions of the human telomerase catalytic subunit essential for in vivo enzyme activity. Mol Cell Biol 22:6234–6246CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Weiss MA, Narayana N (1998) RNA recognition by arginine-rich peptide motifs. Biopolymers 48:167–180. doi: 10.1002/(SICI)1097-0282(1998)48:2<167:AID-BIP6>3.0.CO;2-8 CrossRefPubMedGoogle Scholar
  14. 14.
    Hemmerich P, Bosbach S, von Mikecz A, Krawinkel U (1997) Human ribosomal protein L7 binds RNA with an alpha-helical arginine-rich and lysine-rich domain. Eur J Biochem 245:549–556CrossRefPubMedGoogle Scholar
  15. 15.
    Su L, Radek JT, Hallenga K et al (1997) RNA recognition by a bent α-helix regulates transcriptional antitermination in phage λ. Biochemistry 36:12722–12732. doi: 10.1021/bi971408k CrossRefPubMedGoogle Scholar
  16. 16.
    Frankel AD, Young JA (1998) HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67:1–25. doi: 10.1146/annurev.biochem.67.1.1 CrossRefPubMedGoogle Scholar
  17. 17.
    Smith CA, Chen L, Frankel AD (2000) Using peptides as models of RNA-protein interactions. Methods Enzymol 318:423–438CrossRefPubMedGoogle Scholar
  18. 18.
    Battiste JL, Mao H, Rao NS et al (1996) Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science 273(80):1547–1551. doi: 10.1126/science.273.5281.1547 CrossRefPubMedGoogle Scholar
  19. 19.
    Puglisi JD, Chen L, Blanchard S, Frankel AD (1995) Solution structure of a bovine immunodeficiency virus Tat–TAR peptide-RNA complex. Science 270:1200–1203. doi: 10.1126/science.270.5239.1200 CrossRefPubMedGoogle Scholar
  20. 20.
    Oguro A, Ohtsu T, Svitkin YV et al (2003) RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis. RNA 9:394–407. doi: 10.1261/rna.2161303 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Nicoletti I, Migliorati G, Pagliacci MC et al (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279. doi: 10.1016/0022-1759(91)90198-O CrossRefPubMedGoogle Scholar
  22. 22.
    Piatyszek MA, Kim NW, Weinrich SL et al (1995) Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci 17:1–15. doi: 10.1007/BF00981880 CrossRefGoogle Scholar
  23. 23.
    Cong Y-S, Wright WE, Shay JW (2002) Human telomerase and its regulation. Microbiol Mol Biol Rev 66:407–425. doi: 10.1128/MMBR.66.3.407-425.2002 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Damm K, Hemmann U, Garin-Chesa P et al (2002) A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 20:6958–6968. doi: 10.1093/emboj/20.24.6958 CrossRefGoogle Scholar
  25. 25.
    Hisatake J, Kubota T, Hisatake Y et al (1999) 5,6-trans-16-ene-vitamin D3: a new class of potent inhibitors of proliferation of prostate, breast, and myeloid leukemic cells. Cancer Res 59:4023–4029PubMedGoogle Scholar
  26. 26.
    Naasani I, Seimiya H, Yamori T, Tsuruo T (1999) FJ5002: a potent telomerase inhibitor identified by exploiting the disease-oriented screening program with COMPARE analysis. Cancer Res 59:4004–4011PubMedGoogle Scholar
  27. 27.
    Glukhov AI, Zimnik OV, Gordeev SA, Severin SE (1998) Inhibition of telomerase activity of melanoma cells in vitro by antisense oligonucleotides. Biochem Biophys Res Commun 248:368–371. doi: 10.1006/bbrc.1998.8801 CrossRefPubMedGoogle Scholar
  28. 28.
    Varshney A, Ramakrishnan SK, Sharma A et al (2014) Global expression profile of telomerase-associated genes in HeLa cells. Gene 547:211–217. doi: 10.1016/j.gene.2014.06.018 CrossRefPubMedGoogle Scholar
  29. 29.
    Ramakrishnan SK, Varshney A, Sharma A et al (2014) Expression of targeted ribozyme against telomerase RNA causes altered expression of several other genes in tumor cells. Tumor Biol 35:5539–5550. doi: 10.1007/s13277-014-1729-z CrossRefGoogle Scholar
  30. 30.
    Wan MS, Fell PL, Akhtar S (1998) Synthetic 2′-O-methyl-modified hammerhead ribozymes targeted to the RNA component of telomerase as sequence-specific inhibitors of telomerase activity. Antisense Nucleic Acid Drug Dev 8:309–317. doi: 10.1089/oli.1.1998.8.309 CrossRefPubMedGoogle Scholar
  31. 31.
    Kondo Y, Komata T, Kondo S (2001) Combination therapy of 2-5A antisense against telomerase RNA and cisplatin for malignant gliomas. Int J Oncol 18:1287–1292PubMedGoogle Scholar
  32. 32.
    Pascolo E, Wenz C, Lingner J et al (2002) Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol Chem 277:15566–15572. doi: 10.1074/jbc.M201266200 CrossRefPubMedGoogle Scholar
  33. 33.
    Ward RJ, Autexier C (2005) Pharmacological telomerase inhibition can sensitize drug-resistant and drug-sensitive cells to chemotherapeutic treatment. Mol Pharmacol 68:779–786. doi: 10.1124/mol.105.011494 PubMedGoogle Scholar
  34. 34.
    Barma DK, Elayadi A, Falck JR, Corey DR (2003) Inhibition of telomerase by BIBR 1532 and related analogues. Bioorganic Med Chem Lett 13:1333–1336CrossRefGoogle Scholar
  35. 35.
    Piotrowska K, Kleideiter E, Mürdter TE et al (2005) Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors. Lab Investig. doi: 10.1038/labinvest.3700352 PubMedGoogle Scholar
  36. 36.
    Bilsland AE, Anderson CJ, Fletcher-Monaghan AJ et al (2003) Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase. Oncogene 22:370–380. doi: 10.1038/sj.onc.1206168 CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang Q, Chen G, Peng L et al (2006) Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dual-regulated oncolytic adenovirus. Clin Cancer Res 12:6523–6531. doi: 10.1158/1078-0432.CCR-06-1491 CrossRefPubMedGoogle Scholar
  38. 38.
    Gomez DE, Tejera AM, Olivero OA (1998) Irreversible telomere shortening by 3′-Azido-2′, 3′-dideoxythymidine (AZT) treatment. Biochem Biophys Res Commun 246:107–110. doi: 10.1006/bbrc.1998.8555 CrossRefPubMedGoogle Scholar
  39. 39.
    Bala J, Bhaskar A, Varshney A et al (2011) In vitro selected RNA aptamer recognizing glutathione induces ROS-mediated apoptosis in the human breast cancer cell line MCF 7. RNA Biol 8:101–111. doi: 10.4161/rna.V.I.14116 CrossRefPubMedGoogle Scholar
  40. 40.
    Nieuwlandt D, Wecker M, Gold L (1995) In vitro selection of RNA ligands to substance P. Biochemistry 34:5651–5659. doi: 10.1021/bi00016a041 CrossRefPubMedGoogle Scholar
  41. 41.
    Xu W, Ellington AD (1996) Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope. Proc Natl Acad Sci USA 93:7475–7480. doi: 10.1073/pnas.93.15.7475 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Gilbert BA, Sha M, Wathen ST, Rando RR (1997) RNA aptamers that specifically bind to a K Ras-derived farnesylated peptide. Bioorganic Med Chem 5(6):1115–1122CrossRefGoogle Scholar
  43. 43.
    Ylera F, Lurz R, Erdmann VA, Fürste JP (2002) Selection of RNA aptamers to the Alzheimer’s disease amyloid peptide. Biochem Biophys Res Commun 290:1583–1588. doi: 10.1006/bbrc.2002.6354 CrossRefPubMedGoogle Scholar
  44. 44.
    Chen J-L, Blasco MA, Greider CW (2000) Secondary structure of vertebrate telomerase RNA. Cell 100:503–514. doi: 10.1016/S0092-8674(00)80687-X CrossRefPubMedGoogle Scholar
  45. 45.
    Brown Y, Abraham M, Pearl S et al (2007) A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs. Nucleic Acids Res 35:6280–6289. doi: 10.1093/nar/gkm713 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Lato SM, Boles AR, Ellington AD (1995) In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution. Chem Biol 2:291–303. doi: 10.1016/1074-5521(95)90048-9 CrossRefPubMedGoogle Scholar
  47. 47.
    Berova N, Nakanishi K, Woody R (2000) Circular dichroism : principles and applications. Wiley, New YorkGoogle Scholar
  48. 48.
    Gilligan TJ, Schwarz G (1976) The self-association of adenosine-5′-triphosphate studied by circular dichroism at low ionic strengths. Biophys Chem 4:55–63CrossRefPubMedGoogle Scholar
  49. 49.
    Sundaram P, Kurniawan H, Byrne ME, Wower J (2013) Therapeutic RNA aptamers in clinical trials. Eur J Pharm Sci 48:259–271. doi: 10.1016/j.ejps.2012.10.014 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Akhil Varshney
    • 1
  • Jyoti Bala
    • 1
  • Baby Santosh
    • 1
  • Ashima Bhaskar
    • 1
  • Suresh Kumar
    • 1
    • 2
  • Pramod K. Yadava
    • 1
  1. 1.Applied Molecular Biology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
  2. 2.Molecular Genetics Laboratory, Institute of Cytogenetic and Preventive OncologyIndian Council of Medical ResearchNoidaIndia

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