Targeting a Pneumocystis carinii group I intron with methylphosphonate oligonucleotides: backbone charge is not required for binding or reactivity.
Pneumocystis carinii is a mammalian pathogen that contains a self-splicing group I intron in its large subunit rRNA precursor. We report the binding of methylphosphonate/DNA chimeras and neutral methylphosphonate oligonucleotides to a ribozyme that is a truncated form of the intron. At 15 mM Mg(2+), the nuclease-resistant all-methylphosphonate hexamer, d(AmTmGmAmCm)rU, with a sequence that mimics the 3' end of the precursor's 5' exon, binds with a dissociation constant of 272 nM. The hexamer's dissociation constant for binding by base-pairing alone to the ribozyme's binding site sequence is 8.3 mM. Thus there is a 30 000-fold binding enhancement by tertiary interactions (BETI), which is close to the 60 000-fold enhancement previously observed with the all-ribo hexamer, r(AUGACU). Evidently, backbone charge and 2' hydroxyl groups are not required for BETI. At 3-15 mM Mg(2+), the all-methylphosphonate and DNA oligonucleotides trans-splice to a truncated form of the rRNA precursor, but do not compete with cis-splicing when pG is present. These results suggest that uncharged or partially charged backbones may be used to design therapeutics to target RNAs through binding enhancement by tertiary interactions and suicide inhibition strategies.
Disney
M D
MD
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Testa
S M
SM
Turner
D H
DH
D001665:Binding Sites; D007438:Introns; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011010:Pneumocystis / Q000235:genetics*; D012322:RNA Precursors / Q000737:chemistry; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry; D013237:Stereoisomerism; D013816:Thermodynamics
D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; C032627:methylphosphonic acid