Abstract
The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.
Highlights
systemic evolution of ligands by exponential enrichment (SELEX) technology is a dedicated selection process for generating specific aptamers
Because the changes of aptamer family and round-to-round enrichment can be tracked throughout selection cycles, HT-SELEX is capable of identifying candidate aptamers with high affinity at a much earlier selection round, which can reduce the cost and time associated with over-selection, and avoid potential PCR artifacts in the re-amplification step[28]
We demonstrated that HT-SELEX is a powerful and precise tool for guiding library design and aptamer selection
Summary
SELEX (systemic evolution of ligands by exponential enrichment) technology is a dedicated selection process for generating specific aptamers. Droplet digital PCR (ddPCR) or emulsion PCR (ePCR) has been incorporated into the SELEX protocol to reduce the propagation of byproducts, which putatively avoids PCR bias and selection failure[28,29,30,31] These systems are methods that compartmentalize and miniaturize the PCR reaction by generating a water-in-oil emulsion containing numerous droplets, which creates a local homogeneous amplification micro-environment. A detailed, parallel comparison (e.g., of primary sequences, frequency of aptamer family, total reads, and binding affinity) of the impacts of solution PCR and ddPCR on the RNA aptamer-selection process is needed These studies showed the advantages of ddPCR for aptamer selection, no direct sequence analysis of random DNA libraries generated by ddPCR has been reported. The nucleotide composition in initial RNA libraries has already been studied by other groups[32,33,34], existence of sequence bias has never been discussed
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