Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron.
Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K(d) of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a G.U pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r((___)ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r((__)GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.
Disney
M D
MD
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Gryaznov
S M
SM
Turner
D H
DH
D000818:Animals; D020029:Base Pairing; D001665:Binding Sites / Q000235:genetics; D005091:Exons; D006801:Humans; D007438:Introns; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D018716:Molecular Mimicry; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D011010:Pneumocystis / Q000201:enzymology* / Q000235:genetics; D012322:RNA Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry
D009841:Oligonucleotides; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D013873:Thionucleotides; D008274:Magnesium