Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Abstract

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of "complementary addressed modification titration" (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylamino]benzylamino- group (C1RCH2NH)- at 5'-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E1, E2 and their derivatives E1Phn, E2Phn bearing N-(2-hydroxyethyl)-phenazinium residues Phn- both at 5'- and 3'-ends covalently linked via ethylenediamine linker were used as effectors. Effectors E1 and E2 (E1Phn and E2Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8m) reagents were used for the target modification. The reagent X8m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8m and effectors E1, E2, E1Phn, E2Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group C1RCH2NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either C1RCH2NH- group or TT-mismatch at the junction.

Propagation of Melting Cooperativity along the Phosphodiester Backbone of DNA

“…The effect of base stacking on the cooperativity of DNA duplex formation was investigated before via chemical ligation of a reporter oligonucleotide to its template in the presence and absence of a third oligonucleotide bound adjacently to the template. [2][3][4] It was shown that two pyrimidine bases at the junction site lead to no increase in the binding constant of the reporter oligonucleotide, while a 28-fold enhancement was observed when two guanines are next to each other, indicating a considerable positive cooperative effect for strand association mediated by purine stacking. 4 A similar situation is encountered in DNA triplex formation of two adjacent pyrimidine strands on a DNA duplex template.…”

Effect of Base Stacking on the Relative Thermodynamic Stability of Oligonucleotide Complexes: A Spectroscopic Study

“…A comparative understanding of DNA sequence-dependent thermodynamic stability and solution hybridisation behaviour would have direct practical applications in the design of multiplex reactions (12). In this regard, co-operative parameter interactions at the nick position have previously been calculated using a complementary addressed modification titration (CAMT) method (13)(14)(15). Free energy values of coaxial stacking for a range of possible interaction types at a nick position have also been determined for two-strand systems where a short strand contiguously stacks to a hairpin oligonucleotide (16,19).…”