Structural and kinetic aspects of chemical reactions in DNA duplexes. Information on DNA local structure obtained from chemical ligation data

Abstract

Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.

The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide

“…It may be explained by the side reaction of BrCN with 3'-terminal NH2-group of hexanucleotide (duplex Ic) or by blocking the nucleophilic attack because of BrCN interaction with both phosphate groups facing the nick (duplexes Id and lIc). In contrast, accumulation of the phosphate activated adduct does not occur if CDI is used as a condensing reagent [8].…”

“…Synthesis of d(ACGGAT-NH2) was performed as described earlier [2]. Synthesis of oligomers with changed configuration at C2'or C3' atoms was described in the previous publication [8].…”

“…We showed earlier [2,8] that the efficiency of CDI-induced ligation strongly depends on the nucleophilicity of the reacting groups and on the conformation of the nicked site. To know, whether these dependences remain when BrCN is used, we carried out coupling reactions on duplexes I and II (Scheme 1) in a parallel series experiments using both condensing agents.…”

“…The primary structure of oligonucleotides prepared by the nick ligation in duplexes I and II was confirmed by Maxam-Gilbert analysis, the nature of the resultant unnatural pyrophosphate (duplexes Id and Ih, IIc) or phosphoramidate (duplex Ic) bonds was proved by their selective cleavage, as described in [2]. The nature of the bond formed by rU (duplexes lo,p, Table 1), as well as of dxT (duplexes Ik,l) and aU (duplexes Im,n) was proved by the alkaline and enzymatic hydrolysis of the corresponding oligomers [8].…”

Surface-promoted replication and exponential amplification of DNA analogues

“…3c). Chemical ligation 37,38 was achieved by replacing the hybridization buffer with a ligation buffer containing N-ethyl-N9-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) as the condensing agent. To determine the ef®ciency of ligation, product formation was monitored by the HPLC analysis of the mixture of products obtained after cleaving the disulphide bonds of an aliquot of the templated supports using DTT as the reducing agent (Fig.…”