Sequence Composition Effects on the Energetics of Triple Helix Formation by Oligonucleotides Containing a Designed Mimic of Protonated Cytosine

Abstract

A nonnatural nucleoside, 1- (2-deoxy-β-ribofuranosyl)-3-methyl-5-amilHn-op-y razol0[4,3-d]pyrimidin-7-one (P), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine.purine.pyrimidine triple helix. Quantitative footprint titration experiments at neutral pH (22 °C, 100 mM NaCl, 10 mM bis-tris, 250 μM spermine) reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing P or 5-methylcytosine (^mC). Purine tracts of sequence composition 5'-d(AAAAAGAGAGAGAGA)-3' are bound by oligonucleotide 5'-d (TTTTT^mCT^mCT^mCT^mCT^mCT)-3' 4 orders of magnitude more strongly than by 5'-d (TTTTTPTPTPTPTPT)-3' (K_T ≈ 3 x l0^9 M^(-1) and KT = 1 x l0^5 M^(-1), respectively). Conversely, purine tracts of sequence composition 5'-d(AAAAGAAAAGGGGGGA)-3' are bound by oligonucleotide 5'-d(TTTT^mCTTTT)-3' 5 orders of magnitude less strongly than by 5'-d(TTTT^mCTTTTPPPPPPT)-3' (K_T < 5 x l0^4 M^(-1) and K_T ≈ 4 x l0^9 M^(-1), respectively). The complementary nature of P and ^mC expands the repertoire of G-rich sequences which may be targeted by triple helix formation.