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Journal of Molecular Evolution

, Volume 82, Issue 2–3, pp 81–92 | Cite as

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

  • Lively Lie
  • Shweta Biliya
  • Fredrik Vannberg
  • Roger M. Wartell
Original Article

Abstract

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg2+. Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze–thaw cycle and a maximum of 60 % was obtained after several freeze–thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14–24 % in one freeze–thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

Keywords

Ribozyme RNA ligation RNA world Dehydration 

Notes

Acknowledgments

We thank Rachel Hutto for assistance with ligation experiments and Prof. Klemens Hertel for advice on the carbodiimide reaction. We gratefully acknowledge support during the course of this study from the NASA Astrobiology Institute, and a Dept. of Education GAANN Fellowship awarded to L. Lie.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

239_2016_9729_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1256 kb)

References

  1. Adamala K, Szostak JW (2013) Nonenzymatic template-directed RNA synthesis inside model protocells. Science 342:1098CrossRefPubMedPubMedCentralGoogle Scholar
  2. Anderson M, Schultz EP, Martick M, Scott WG (2013) Active-site monovalent cations revealed in a 1.55-angstrom-resolution hammerhead ribozyme structure. J Mol Biol 425:3790CrossRefPubMedPubMedCentralGoogle Scholar
  3. Attwater J, Wochner A, Pinheiro VB, Coulson A, Holliger P (2010) Ice as a protocellular medium for RNA replication. Nat Commun 1:76CrossRefPubMedGoogle Scholar
  4. Auffinger P, Grover N, Westhof E (2011) Metal ion binding to RNA. Met Ions Life Sci 9:1CrossRefPubMedGoogle Scholar
  5. Bada JL, Lazcano A (2002) Origin of life. Some like it hot, but not the first biomolecules. Science 296:1982CrossRefPubMedGoogle Scholar
  6. Biondi E, Maxwell AW, Burke DH (2012) A small ribozyme with dual-site kinase activity. Nucleic Acids Res 40:7528CrossRefPubMedPubMedCentralGoogle Scholar
  7. Birikh KR, Heaton PA, Eckstein F (1997) The structure, function and application of the hammerhead ribozyme. Eur J Biochem 245:1CrossRefPubMedGoogle Scholar
  8. Canny MD, Jucker FM, Kellogg E, Khvorova A, Jayasena SD, Pardi A (2004) Fast cleavage kinetics of a natural hammerhead ribozyme. J Am Chem Soc 126:10848CrossRefPubMedGoogle Scholar
  9. Canny MD, Jucker FM, Pardi A (2007) Efficient ligation of the Schistosoma hammerhead ribozyme. Biochemistry 46:3826CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chi YI, Martick M, Lares M, Kim R, Scott WG, Kim SH (2008) Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biol 6:2060Google Scholar
  11. Collins KD (2006) Ion hydration: implications for cellular function, polyelectrolytes, and protein crystallization. Biophys Chem 119:271CrossRefPubMedGoogle Scholar
  12. Darnell J (2011) RNA: life’s indispensable molecule. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  13. de la Pena M, Garcia-Robles I (2010) Ubiquitous presence of the hammerhead ribozyme motif along the tree of life. RNA 16:1943CrossRefPubMedPubMedCentralGoogle Scholar
  14. De la Pena M, Gago S, Flores R (2003) Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity. EMBO J 22:5561CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ditzler MA, Lange MJ, Bose D, Bottoms CA, Virkler KF, Sawyer AW, Whatley AS, Spollen W, Givan SA, Burke DH (2013) High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase. Nucleic Acids Res 41:1873CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fedor MJ (2009) Comparative Enzymology and Structural Biology of RNA Self-Cleavage. Annu Rev Biophys 38:271CrossRefPubMedGoogle Scholar
  17. Ferris JP, Hill AR Jr, Liu R, Orgel LE (1996) Synthesis of long prebiotic oligomers on mineral surfaces. Nature 381:59CrossRefPubMedGoogle Scholar
  18. Franks F (2000) Water a matrix of life. Royal Society of Chemistry, Cambridge xii, 225 Google Scholar
  19. Gao JY, Xie C, Wang YL, Xu Z, Hao HX (2012) Solubility data of trisodium citrate hydrates in aqueous solution and crystal-solution interfacial energy of the pentahydrate. Cryst Res Technol 47:397CrossRefGoogle Scholar
  20. Gilbert W (1986) Origin of Life—the Rna World. Nature 319:618CrossRefGoogle Scholar
  21. Hammann C, Luptak A, Perreault J, de la Pena M (2012) The ubiquitous hammerhead ribozyme. RNA 18:871CrossRefPubMedPubMedCentralGoogle Scholar
  22. Han J, Burke JM (2005) Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site. Biochemistry 44:7864CrossRefPubMedGoogle Scholar
  23. Haynes CM (2013) The CRC handbook of chemistry and physics. Libr J 94:192Google Scholar
  24. Hertel KJ, Herschlag D, Uhlenbeck OC (1994) A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry 33:3374CrossRefPubMedGoogle Scholar
  25. Hertel KJ, Herschlag D, Uhlenbeck OC (1996) Specificity of hammerhead ribozyme cleavage. EMBO J 15:3751PubMedPubMedCentralGoogle Scholar
  26. Higashi K, Terui Y, Suganami A, Tamura Y, Nishimura K, Kashiwagi K, Igarashi K (2008) Selective structural change by spermidine in the bulged-out region of double-stranded RNA and its effect on RNA function. J Biol Chem 283:32989CrossRefPubMedGoogle Scholar
  27. Hsiao C, Chou IC, Okafor CD, Bowman JC, O’Neill EB, Athavale SS, Petrov AS, Hud NV, Wartell RM, Harvey SC, Williams LD (2013) RNA with iron(II) as a cofactor catalyses electron transfer. Nat Chem 5:525CrossRefPubMedGoogle Scholar
  28. Hutchins CJ, Rathjen PD, Forster AC, Symons RH (1986) Self-cleavage of plus and minus Rna transcripts of avocado sunblotch viroid. Nucleic Acids Res 14:3627CrossRefPubMedPubMedCentralGoogle Scholar
  29. Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP (2001) RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science 292:1319CrossRefPubMedGoogle Scholar
  30. Joyce GF (1989) RNA evolution and the origins of life. Nature 338:217CrossRefPubMedGoogle Scholar
  31. Kanavarioti A, Monnard PA, Deamer DW (2001) Eutectic phases in ice facilitate nonenzymatic nucleic acid synthesis. Astrobiology 1:271CrossRefPubMedGoogle Scholar
  32. Kazakov SA, Balatskaya SV, Johnston BH (1998) Freezing-induced self-ligation of the hairpin ribozyme: cationic effects. Struct Motion Interact Expr Biol Macromol 2:155Google Scholar
  33. Kazakov SA, Balatskaya SV, Johnston BH (2006) Ligation of the hairpin ribozyme in cis induced by freezing and dehydration. Rna 12:446CrossRefPubMedPubMedCentralGoogle Scholar
  34. Khvorova A, Lescoute A, Westhof E, Jayasena SD (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat Struct Biol 10:708CrossRefPubMedGoogle Scholar
  35. Kupakuwana GV, Crill JE 2nd, McPike MP, Borer PN (2011) Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing. PLoS One 6:e19395CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lambert D, Draper DE (2007) Effects of osmolytes on RNA secondary and tertiary structure stabilities and RNA-Mg2+ interactions. J Mol Biol 370:993CrossRefPubMedPubMedCentralGoogle Scholar
  37. Levy M, Miller SL (1998) The stability of the RNA bases: implications for the origin of life. Proc Natl Acad Sci USA 95:7933CrossRefPubMedPubMedCentralGoogle Scholar
  38. Li YF, Breaker RR (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2 ‘-hydroxyl group. J Am Chem Soc 121:5364CrossRefGoogle Scholar
  39. Murray JB, Seyhan AA, Walter NG, Burke JM, Scott WG (1998) The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol 5:587CrossRefPubMedGoogle Scholar
  40. Mutschler H, Holliger P (2014) Non-canonical 3′-5′ extension of RNA with prebiotically plausible ribonucleoside 2′,3′-cyclic phosphates. J Am Chem Soc 136:5193CrossRefPubMedPubMedCentralGoogle Scholar
  41. Mutschler H, Wochner A, Holliger P (2015) Freeze-thaw cycles as drivers of complex ribozyme assembly. Nat Chem 7:502CrossRefPubMedPubMedCentralGoogle Scholar
  42. O’Rear JL, Wang SL, Feig AL, Beigelman L, Uhlenbeck OC, Herschlag D (2001) Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations. Rna 7:537CrossRefPubMedPubMedCentralGoogle Scholar
  43. Pace NR (1991) Origin of life–facing up to the physical setting. Cell 65:531CrossRefPubMedGoogle Scholar
  44. Perreault J, Weinberg Z, Roth A, Popescu O, Chartrand P, Ferbeyre G, Breaker RR (2011) Identification of hammerhead ribozymes in all domains of life reveals novel structural variations. PLoS Comput Biol 7:e1002031CrossRefPubMedPubMedCentralGoogle Scholar
  45. Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G (1986) Autolytic processing of dimeric plant-virus satellite Rna. Science 231:1577CrossRefPubMedGoogle Scholar
  46. Renz M, Lohrmann R, Orgel LE (1971) Catalysts for polymerization of adenosine cyclic 2′,3′-phosphate on a poly (U) template. Biochim Biophys Acta 240:463CrossRefPubMedGoogle Scholar
  47. Roy S (2008) Cleavage and ligation studies in hairpin and hammerhead ribozymes using site specific nucleotide modifications. University of Vermont, Burlington, p 176Google Scholar
  48. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  49. Serganov A, Patel DJ (2009) Amino acid recognition and gene regulation by riboswitches. Biochim Biophys Acta Gene Regul Mech 1789:592CrossRefGoogle Scholar
  50. Shepotinovskaya IV, Uhlenbeck OC (2008) Catalytic diversity of extended hammerhead ribozymes. Biochemistry 47:7034CrossRefPubMedPubMedCentralGoogle Scholar
  51. Stage-Zimmermann TK, Uhlenbeck OC (1998) Hammerhead ribozyme kinetics. RNA 4:875CrossRefPubMedPubMedCentralGoogle Scholar
  52. Stage-Zimmermann TK, Uhlenbeck OC (2001) A covalent crosslink converts the hammerhead ribozyme from a ribonuclease to an RNA ligase. Nat Struct Biol 8:863CrossRefPubMedGoogle Scholar
  53. Sun X, Li JM, Wartell RM (2007) Conversion of stable RNA hairpin to a metastable dimer in frozen solution. RNA 13:2277CrossRefPubMedPubMedCentralGoogle Scholar
  54. Tokumoto Y, Saigo K (1992) RNA-RNA and RNA-DNA ligation with the sTobRV(+) hammerhead ribozyme. Nucleic Acids Symp Ser 27:21PubMedGoogle Scholar
  55. Trinks H, Schroder W, Biebricher CK (2005) Ice and the origin of life. Orig Life Evol Biosph 35:429CrossRefPubMedGoogle Scholar
  56. Turk RM, Chumachenko NV, Yarus M (2010) Multiple translational products from a five-nucleotide ribozyme. Proc Natl Acad Sci USA 107:4585CrossRefPubMedPubMedCentralGoogle Scholar
  57. Uhlenbeck OC (1987) A small catalytic oligoribonucleotide. Nature 328:596CrossRefPubMedGoogle Scholar
  58. Vlassov AV, Johnston BH, Landweber LF, Kazakov SA (2004) Ligation activity of fragmented ribozymes in frozen solution: implications for the RNA world. Nucleic Acids Res 32:2966CrossRefPubMedPubMedCentralGoogle Scholar
  59. Vlassov AV, Kazakov SA, Johnston BH, Landweber LF (2005) The RNA world on Ice: a new scenario for the emergence of RNA information. J Mol Evol 61:264CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.School of BiologyGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Parker H. Petit Institute for Bioengineering and BioscienceGeorgia Institute of TechnologyAtlantaUSA

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