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Assembly of complex RNAs by splinted ligation.

Authors
  • Akiyama, Benjamin M1
  • Stone, Michael D
  • 1 Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California, USA.
Type
Published Article
Journal
Methods in enzymology
Publication Date
Jan 01, 2009
Volume
469
Pages
27–46
Identifiers
DOI: 10.1016/S0076-6879(09)69002-9
PMID: 20946783
Source
Medline
License
Unknown

Abstract

Mechanistic studies of RNA enzymes (ribozymes) and ribonucleoprotein (RNP) complexes such as the ribosome and telomerase, often seek to characterize RNA structural features, either dynamic or static, and relate these properties to specific catalytic functions. Many experimental techniques that probe RNA structure-function relationships rely upon site-specific incorporation of chemically modified ribonucleotides into the RNA of interest, often in the form of chemical cross-linkers to probe for sites of protein-RNA interaction or small organic fluorophores to measure dynamic structural properties of RNAs. The ability to arbitrarily modify any RNA molecule has been greatly enabled by modern RNA synthesis techniques; however, there remains a practical size limitation (~70 bases). Consequently, experimental approaches involving specific chemical modifications of larger RNAs require the use of RNA ligation methods. The aim of this chapter is to describe a general approach for covalently joining multiple site-specifically modified RNA fragments, drawing from our fluorescence-based structural studies of telomerase RNA as an example.

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