In vitro selection of bacteriophage φ29 prohead RNA aptamers for prohead binding

Prohead RNA (pRNA) of the Bacillus subtilis bacteriophage φ29 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 wildtype. 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 of pRNA.