CD and Melting Curves Structural Studies of the Tandem DNA Complex Formed with Oligonucleotides Carrying Photoactive and Sensitizing Groups in the Nick Region

Abstract

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC)and two 7-mer oligonucleotide conjugates,one of which was modified on its 3'-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group,and the other one was linked through its 5'-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD)spectra were detected for all possible oligonucleotide and/or conjugate combinations.In addition,molecular modeling simulation of the complex structure was carried out. It was found that CD spectra did not show serious changes in the B-helix structure of the duplex. The interaction between perylene-and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner,but azido-group located on the side of DNA chain minor groove.