Substrate efficiency of CY5-modified derivatives of deoxyuridine and deoxycytidine in the rolling circle amplification

The kinetics of amplification and the features of individual and simultaneous incorporation of modified deoxynucleoside triphosphates in DNA during rolling circle amplification (RCA) have been studied. The study was carried out for six pairs of Sy5-labeled triphosphates of deoxyuridine (dU) and deoxycytidine (dC) previously synthesized with similar fluorescent substituents inside the pair. The effect of the linker length between the fluorophore and the pyrimidine base on the incorporation density was determined: nucleotides with a linker length of six carbon atoms are embedded in a growing DNA chain better than with three carbon atoms. It was found that the combined introduction of triphosphates into the reaction in an equivalent total concentration does not enhance the inhibitory effect, which gives grounds for a more detailed study of the simultaneous use of labeled dU and dC.