Mapping targetable sites on human telomerase RNA pseudoknot/template domain using 2'-OMe RNA-interacting polynucleotide (RIPtide) microarrays

J Biol Chem. 2012 May 25;287(22):18843-53. doi: 10.1074/jbc.M111.316596. Epub 2012 Mar 26.

Abstract

Most cellular RNAs engage in intrastrand base-pairing that gives rise to complex three-dimensional folds. This self-pairing presents an impediment toward binding of the RNA by nucleic acid-based ligands. An important step in the discovery of RNA-targeting ligands is therefore to identify those regions in a folded RNA that are accessible toward the nucleic acid-based ligand. Because the folding of RNA targets can involve interactions between nonadjacent regions and employ both Watson-Crick and non-Watson-Crick base-pairing, screening of candidate binder ensembles is typically necessary. Microarray-based screening approaches have shown great promise in this regard and have suggested that achieving complete sequence coverage would be a valuable attribute of a next generation system. Here, we report a custom microarray displaying a library of RNA-interacting polynucleotides comprising all possible 2'-OMe RNA sequences from 4- to 8-nucleotides in length. We demonstrate the utility of this array in identifying RNA-interacting polynucleotides that bind tightly and specifically to the highly conserved, functionally essential template/pseudoknot domain of human telomerase RNA and that inhibit telomerase function in vitro.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Humans
  • Nucleic Acid Conformation
  • Oligonucleotide Array Sequence Analysis*
  • RNA / chemistry*
  • RNA / genetics*
  • Telomerase / genetics
  • Telomerase / metabolism*

Substances

  • RNA
  • Telomerase