In vitro selection of bacteriophage phi29 prohead RNA aptamers for prohead binding.

The Journal of Biological Chemistry
F Zhang, D L Anderson

Abstract

Prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi29 is needed for in vitro packaging of DNA-gene product 3 (DNA-gp3). Residues 22-84 of the 174-base pRNA bind the portal vertex of the prohead, the site of DNA packaging. To define the nucleotides of pRNA needed for prohead binding and DNA-gp3 packaging and to seek biologically active variants of pRNA, segments of pRNA were randomized to obtain vast repertoires of RNA molecules. RNA aptamers, ligands best suited for prohead binding, were obtained by multiple rounds of in vitro selection. Evolution of pRNA aptamers was followed by a competition binding assay and nucleotide sequencing, and mutants were tested for DNA-gp3 packaging. Aptamers selected following randomization of the E stem and loop and a part of the C-E loop that were active in DNA-gp3 packaging were invariably wild-type. DNA-gp3 packaging activity also required nucleotides G82 and G83 that form base pairs intermolecularly with C47 and C48 to produce a novel hexameric oligomer of pRNA. The only mutant aptamers that retained full DNA-gp3 packaging activity showed changes of the U residues at positions 81, 84, and 85 of the D loop. Thus, the in vitro selections essentially recapitulated the natural evolution...Continue Reading

References

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Citations

Oct 31, 2006·Analytical and Bioanalytical Chemistry·Subash Chandra Bose Gopinath
Jul 21, 2010·Analytical and Bioanalytical Chemistry·Jing ZhouYong Wang
Sep 14, 2007·Archives of Virology·S C B Gopinath
Nov 30, 2002·Biochimie·Nancy Bourassa, François Major
Jan 25, 2006·Journal of Nanoscience and Nanotechnology·Peixuan Guo
Apr 9, 2009·Acta Biochimica Et Biophysica Sinica·Chenglong WangHongmei Ding
Oct 26, 2001·Journal of Molecular Biology·C WagnerC Brunel
Jun 17, 2010·Biochemistry·Steven Harris, Susan J Schroeder

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