A Comparative Behavior Analysis of Cy5-Pyrimidine Nucleotides in Rolling Circle Amplification

Objective: The introduction of fluorescently labeled nucleotides in the process of DNA amplification in molecular genetic analysis is a very attractive alternative to post-labeling or the use of labeled primers in the reaction (RCA). Methods: To study the substrate efficiency, the kinetic index (amplification efficiency) was studied, which allowed us to evaluate the inhibitory effect of modified substrates; as well as the yield of the isothermal amplification reaction product and the density of embedding the label into the growing DNA chain. Results and Discussion: Two pairs of Cy5-labeled dU and dC triphosphates with similar electroneutral fluorophore structures, differing in the length of the hydrocarbon linker between the fluorophore and the nitrogenous base were synthesized. A comparative analysis of their substrate behavior in the rolling circle amplification (RCA) reaction using Bst 3.0 DNA polymerase was carried out. It was found that nucleotides with a long linker between the fluorophore and the pyrimidine base are more efficiently incorporated into the growing DNA chain, while the nucleotides with a short linker cause lesser inhibition of RCA. In each of the pairs consisting of dU and dC with similar fluorophores and linkers, the fluorescently labeled uridine derivatives demonstrated a higher incorporation density. It was revealed that, under simultaneous incorporation of the labeled dU and dC, the inhibitory effects do not sum up. This gives grounds for a more careful study of different Cy5-dC variants with a view to increasing the sensitivity of the analysis under simultaneous introduction of the labeled dU and dC. Conclusions: The work presents the results of comparative study of the substrate properties of fluorescently labeled deoxynucleoside triphosphates in RCA. The influence of the chemical nature of the nucleotide (dU or dC) and the structure of the fluorophore on the substrate properties of modified deoxynucleoside triphosphates has been studied. Deoxyuridines with a long linker between the fluorophore and the nitrogenous base were found to be the most effective substrates. In addition, the possibility of simultaneous use of labeled dU and dC in the process of isothermal amplification has been shown, which is potentially capable of increasing the sensitivity of the analysis method.