Evaluation of the Diversity of Random DNA-Libraries by the Shape of Amplification Curves for Estimation of the Efficiency of Aptamer Selection

• Published in: Biochemistry (Moscow). Supplement. Series B, Biomedical chemistry Biomedical chemistry, 2020-04, Vol.14 (2), p.159-167
• Main Authors: Radko, S. P., Lapa, S. A., Chudinov, A. V., Khmeleva, S. A., Mannanova, M. M., Kurbatov, L. K., Kiseleva, Y. Y., Zasedatelev, A. S., Lisitsa, A. V.
• Format: Article
• Language: English
• Subjects: Aptamers; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Deoxyribonucleic acid; DNA; Medicinal Chemistry; Nucleotide sequence; Oligonucleotides; Polymerase chain reaction; Smad protein; Smad4 protein
• ISSN: 1990-7508; 1990-7516
• DOI: 10.1134/S1990750820020109

— Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within the interval from 1 to 10 4 and remained practically unaltered when the number of variants was in the interval from 10 5 to 10 8 . The obtained dependence was used in the course of the SELEX procedure to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.