Inhibition of phage φ29 assembly by antisense oligonucleotides targeting viral pRNA essential for DNA packaging

Abstract

A sensitive and efficient system for the functional assay of antisense oligonucleotides (oligos) was developed based on an in vitro viral assembly system. A 120-base RNA (pRNA), which indispensably participates in bacteriophage φ29 DNA packaging, was the target for antisense action. Antisense oligos bound to pRNA, as revealed by a slower electrophoretic mobility of pRNA/oligo complexes in comparison with native pRNA. infectious viruses were assembled in vitro with synthetic pRNA and DNA, as well as with viral proteins produced from cloned genes. Up to 107 plaque-forming units per milliliter were obtained in the absence of antisense oligos, while as few as zero plaques were detected in the presence of certain antisense oligos. A 1-base mismatch greatly influenced the inhibitory effect of the antisense oligos, but this 1-based mismatch was not important when the mismatch was placed at the end of the oligo. Five oligos did not bind pRNA or inhibit the assembly of the virion, suggesting that the RNA sequences complementary to these oligos are nonessential or buried internally in the RNA. Viral assembly was strongly inhibited by antisense oligos P15 and P10, targeting either the 5'- or the 3'-end of the pRNA, respectively. Viral assembly was also strongly inhibited by oligo Re, targeting an internal region, residues 75-91, of pRNA. Oligo P6 inhibited DNA packaging activity by blocking the binding of pRNA to the procapsid, while P10 and P15 inhibited DNA packaging activity but did not block the binding of pRNA to the procapsid, suggesting that in addition to the reported internal domain for procapsid binding, pRNA contains another domain at the paired 5'/3'-ends with a yet to be defined role in DNA translocation. © 1995 Academic Press, Inc.