Abstract
BackgroundAptamers, single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). In SELEX, monitoring the enriching statuses of aptamer candidates during the process is a key step until today. Conformational change of an aptamer caused by target-binding in gel can be used to indicate its statuses of binding.ResultsIn this study, an easy-to-implement gel-based diffusion method (GBDM) was developed to monitor the interaction between enriched aptamer candidates and their targets. In order to prove the concept, characterization of aptamers targeting their targets including protein (thrombin) and non-protein molecules (acetamiprid, ATP, atrazine, profenofos and roxithromycin), respectively, were performed using mini gels. Our method has advantages over the common methods including easy performed with labor- and time- saving in experimental operation. The concept has been proven by monitoring enrichment of dynamic aptamer candidate libraries targeting a small molecule 2,2-bis(4-chlorophenyl) acetic acid (DDA) during SELEX process. A mini gel cassette was designed and fabricated by our laboratory to make mini agarose gels for diffusion with different directions.ConclusionsThese results indicate that GBDM, in particular, chasing diffusion is suitable for monitoring the interaction between enriched aptamer candidates and their targets. These pioneering efforts are helpful for novel aptamer selection by breaking through the technical bottleneck of aptamer development and helpful for development of novel aptasensors.
Highlights
Aptamers, single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX)
We have evaluated a range of approaches including the methods of electrophoretic mobility shift assay (EMSA) [34], dot blotting, Eastern blotting and target-capture assay [51], quartz crystal microbalance (QCM) analysis [52], quantitative PCR (qPCR)
One of gel-based diffusion method (GBDM), is the most practically useful to monitor the interaction between enriched aptamer candidates and their targets round by round during SELEX process
Summary
Single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is a common method currently used for isolating high-affinity single-stranded (ss) DNAs or RNAs from a large library with random sequences [1,2,3]. These SELEX-derived DNAs and RNAs named aptamers, can be selected against a broad range of targets (e.g. proteins, cells, virus, microorganisms, toxins and chemical compounds) [4,5,6]. More highly target analytes-specific aptamers need to be selected
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