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

Abstract

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.

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma

“…SMAD4 is a common partner for proteins of the RSMAD (receptor-regulated SMADs) group involved in the formation of transcriptionally active complexes induced by transforming growth factor beta [39]. Since SMAD4 is considered as a potential target for targeted cancer therapies [39], we recently tried to select DNA aptamers against recombinant SMAD4 protein (rSMAD4) by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) method [30]. As a result, oligonucleotides containing 5'-GTCT-3' and 5'-AGAC-3' sequences known as SMAD-binding elements (SBE) have evolved by the end of selection, thus indicating that the SMAD4-SBE interaction dominated the aptamer selection process.…”

“…As a result, oligonucleotides containing 5'-GTCT-3' and 5'-AGAC-3' sequences known as SMAD-binding elements (SBE) have evolved by the end of selection, thus indicating that the SMAD4-SBE interaction dominated the aptamer selection process. The SBE sequences were used to construct the anti-SMAD4 aptamer [30], as shown in Table 1. The equilibrium dissociation constant, K d , of the aptamer/SMAD4 complexes, measured by microscale thermophoresis, was found to equal (45 ± 7) nM.…”

“…Though SMAD4 is not considered as a potential circulating biomarker for cancer diagnostics, this is an intracellular protein that is not normally present in blood plasma at a detectable level and therefore will not interfere with the detection of rSMAD4 spiked in plasma. Moreover, the immobilization of the anti-SMAD4 aptamer via the biotin moiety (Table 1) on streptavidin-coated magnetic beads was shown to allow the selective pull down of rSMAD4 from E. coli cell lysates as demonstrated by electrophoretic analysis [30]. Thus, rSMAD4 and the anti-SMAD4 aptamer were employed here as a convenient model for evaluating aptamers as affinity reagents for the enhancement of SRM-based detection of low-abundance proteins in blood plasma.…”

“…Protein concentrations were measured with the BCA assay (Thermo Fisher Scientific #23235). More details on the expression, purification, and characterization of rSMAD4 may be found in [30].…”

“…In the present work, using recombinant protein SMAD4 (mothers against decapentaplegic homolog 4, the neXtProt accession number NX_Q13485) as a model target added at known concentrations to undepleted human blood plasma samples, we investigated the relationship between the initial amount of the target protein and its amount in the enriched fraction, as determined by SRM. The DNA aptamer constructed on the basis of the consensus sequence recognized by SMAD4 [30] and immobilized on magnetic beads was used for the aptamer-based enrichment of the target protein. It was found that aptamer-based enrichment can enhance the sensitivity of SRM detection of recombinant SMAD4 (rSMAD4) by a factor of 18 to 26, depending on the protein concentration in blood plasma, that results in about a 30-fold decrease of the limit of detection.…”