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Nucleic Acids Research Advance Access published online on February 5, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1085
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

Elzbieta Kierzek1,2, Ryszard Kierzek2, Walter N. Moss1, Shawn M. Christensen3,4, Thomas H. Eickbush3 and Douglas H. Turner1,5,*

1Department of Chemistry, University of Rochester, RC Box 270216, Rochester, NY 14627-0216, USA, 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-714 Poznan, Noskowskiego 12/14, Poland, 3Department of Biology, University of Rochester, RC Box 270211, Rochester, NY 14627, USA, 4Department of Biology, University of Texas at Arlington, Arlington, TX 76019 and 5Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA

*To whom correspondence should be addressed. Tel: +1 585 275 3207; Fax: +1 585 276 0205; Email: turner{at}chem.rochester.edu

Received September 10, 2007. Revised November 16, 2007. Accepted November 19, 2007.

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2' oxygen and 4' carbon of ribose) and 2,6-diaminopurine were incorporated into 2'-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5' coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5' RNA). This R2Bm 5' RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5' RNA.


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