Thermodynamic and Structural Features of Cooperative Interactions in Tandem Oligonucleotide Derivatives Arranged at the Complementary Template. Chemical Modification Data

Abstract

General equations are derived for the limit yield [PZ]∞, of the intraduplex reaction between reactive oligonucleotide derivative X bearing p-(N-2-chloroethyl-N-methyl-amino)phenyl residue and oligonucleotide target P encompassing the sequence complementary to X in the presence of one or two oligonucleotide effectors E₁ and E₂. The latters form the complementary tandem sequence E₁-X-E₂ at the target. It is shown that association constants characterizing the affinity of the reagentX to the effector containing complexes PE₁ PE₂ and PE₁E₂ may be calculated from the dependencies of [PZ]∞, on the initial concentrationx₀ , of X provid- ing the sufficient excess of effectors is present. The approach was applied to reaction of ClRCH₂NHpd(TTCCCA) with 26-mer dTTGCCTTGAATGGGAAGAGGGTCATT and effectors Phn-L-pd(TTCAAGG-C)p-L-Phn (E₁) and Phn-L-pd(TGACCCTC)p-L-Phn (E₂) where Phn- is N-(2-hydroxyethyl)-phenazinium residue and L is -NHCH₂CH₂NH- spacer. The association constants were found to be K _xe1 = 6.75 · 10⁵M⁻¹, K _xe2 = 4.15 · 10⁴M⁻¹ and K _xe12 = 5.87 · 10⁶M⁻¹ as compared with the affinity of X to P K _x = 2.16 · 10⁴M⁻¹ in the absence of effectors. The experiments on self-alkylation of target reactive derivative CIRCH₂NHpd(TTGCCTrGAATGGGAAGAGG- GTCATT) both in the presence and in the absence of effector E₂ as well as the Molecular Mechanics calculations of its prereactive states showed target to form the hairpin secondary structure. Under reasonable suggestions taking into account the internal structure of the target co-operativity parameters describing the contribution of interactions of the terminal nucleotides ofX with adjacent residues of effector were calculated and found to be α₁ = 16, α₂= 10 and α₁₂ = 139 for the duplexes PXE₁ PXE₂ and PXE₁E₂, respectively.