RNA-Directed Recombination of RNA In Vitro

  • Niles Lehman
  • Nilesh Vaidya
  • Jessica A. M. Yeates
Part of the Methods in Molecular Biology book series (MIMB, volume 1240)


Construction of long RNAs can be achieved in vitro by using ribozymes to recombine shorter RNAs. This can be a useful technique to prepare RNAs when the final product is either very long or contains chemical modifications that are difficult to incorporate using standard in vitro transcription techniques. Here, we describe the use of the Azoarcus group I intron ribozyme to recombine shorter RNAs into longer ones. This ribozyme is a generalized RNA recombinase ribozyme that operates rapidly and with high efficiency.

Key words

Electrophoresis Polymerase chain reaction Recombination Ribozyme Group I intron Azoarcus ribozyme Transcription 



We would like to thank A. Burton and B. Larson for help during the preparation of this manuscript. This work was supported by the NASA grant NNX10AR15G to N.L.


  1. 1.
    Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucletide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Rio DC, Ares M Jr, Hannon GJ, Nilsen TW (2011) RNA: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
  3. 3.
    Riley CA, Lehman N (2003) Generalized RNA-directed recombination of RNA. Chem Biol 10:1233–1243PubMedCrossRefGoogle Scholar
  4. 4.
    Lehman N, Unrau PJ (2005) Recombination during in vitro evolution. J Mol Evol 61:245–252PubMedCrossRefGoogle Scholar
  5. 5.
    Reinhold-Hurek B, Shub DA (1992) Self-splicing introns in tRNA genes of widely divergent bacteria. Nature 357:173–176PubMedCrossRefGoogle Scholar
  6. 6.
    Hayden EJ, Riley CA, Burton AS, Lehman N (2005) RNA-directed construction of structurally complex and active ligase ribozymes through recombination. RNA 11:1678–1687PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Draper WE, Hayden EJ, Lehman N (2008) Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides. Nucleic Acids Res 36:520–531PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Burton AS, Madix RA, Vaidya N, Riley CA, Hayden EJ, Chepetan A, Arenas CD, Larson BC, Lehman N (2009) Gel purification of radiolabeled nucleic acids via phosphorimaging: Dip-N-Dot. Anal Biochem 388:351–352PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Niles Lehman
    • 1
  • Nilesh Vaidya
    • 2
  • Jessica A. M. Yeates
    • 1
  1. 1.Department of ChemistryPortland State UniversityPortlandUSA
  2. 2.Department of Chemical and Biological EngineeringPrinceton UniversityPrincetonUSA

Personalised recommendations