Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection

   
Radko S.P.1 , Lapa S.A.2, Chudinov A.V.2, Khmeleva S.A.3, Mannanova M.M.3, Kurbatov L.K.3, Kiseleva Y.Y.4, Zasedatelev A.S.5, Lisitsa A.V.3

1. IBMC-EcoBioPharm Ltd., Moscow, Russia; Institute of Biomedical Chemistry, Moscow, Russia
2. IBMC-EcoBioPharm Ltd., Moscow, Russia; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
3. Institute of Biomedical Chemistry, Moscow, Russia
4. Russian Scientific Center of Roentgenoradiology, Moscow, Russia
5. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Section: Experimental Study
DOI: 10.18097/PBMC20196506477      PubMed Id: 31876518
Year: 2019  Volume: 65  Issue: 6  Pages: 477-484
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 in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX 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.
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Keywords: combinatorial libraries, amplification curves, aptamers, SELEX
Citation:

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. (2019). Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection. Biomeditsinskaya khimiya, 65(6), 477-484.
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