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 immunoc ompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor o f 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 ribo zyme derived from the intron with a K-d Of 5.2 nM, which is 61000-fold tigh ter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., G igliotti, F., and Turner, D. H. (1997) Biochemistry, 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize i nteractions that give rise to this tertiary stability, binding to the riboz yme 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 expecte d 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 potentiall y form a C-G pair with the target has little effect on binding affinity. Tr uncated oligonucleotides were tested for their ability to inhibit intron se lf-splicing via a suicide inhibition mechanism. The tetramer, r(_ _GACU), r etains similar binding affinity and reactivity as the hexamer, r(AUGACU). T hus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results w ith a phosphoramidate tetramer and thiophosphoramidate hexamer indicate tha t oligonucleotides with backbones stable to nuclease digestion retain favor able binding and reactivity properties.