Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Abstract

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

Aptamers as targeted therapeutics: current potential and challenges

“…However, high throughput sequencing (HTS) technology has revolutionized the selection of aptamers by making the selection process visible with each round of selection. HTS and bioinformatics analysis combined with SELEX (HT-SELEX) not only facilitates the rapid identification of high-affinity aptamers, but also reveals a comprehensive landscape for the molecular evolution events 59–61 . Millions of sequence reads can be processed from each selection cycle, thus providing insight into the entire process, including primary sequences, total reads, nucleotide composition, frequency, and rate of molecular enrichment 62, 63 .…”

“…Because HT-SELEX allows quantitative assessment of the dynamic changes in the library composition throughout selection cycles, it is capable of identifying high-affinity aptamers at a much earlier round, which is more cost-efficient and avoids the potential PCR bias associated with over-selection. Global analysis of large sequence data sets by robust bioinformatics tools can further facilitate high throughput characterization of aptamers, including structure prediction, binding affinity/specificity, functional properties, and aptamer–target interactions 59 .…”

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

“…They tested the binding of 10 aptamers with a higher ratio between the two cell lines and identified four aptamers with a higher binding on c-kit expressing cells. A similar strategy was also used by Levay et al where a parallel selection was performed after three rounds in order to identify aptamers against hIL-10RA [55]. Noticeably, they also performed and obtained results in another parallel selection to find aptamers that can bind to both the human and the murine form of IL-10RA.…”

DNA aptamers selection for breast cancer

“…При селекции аптамеров более эффективной в сравнении с классической открытой ПЦР является ПЦР в эмульсии [21][22][23]. При большом разнообразии олигонуклеотидов первыми амплифицируются последовательности с наиболее простой третичной структурой, более легкодоступной для фермента, что приводит к снижению количества возможных кандидатов в аптамеры за счёт сложности их структуры и повышенного содержания G-и C-нуклеотидов [23], особенно на первых раундах селекции. На практике это выглядит как потеря на геле полосы, характерной для пула аптамеров, и снижение уровня связывания аптамеров с мишенью.…”

“…На практике это выглядит как потеря на геле полосы, характерной для пула аптамеров, и снижение уровня связывания аптамеров с мишенью. При использовании ПЦР в эмульсиях или правильно подобранных условиях открытой ПЦР основные кандидаты в аптамеры восстанавливаются после третьего раунда [23]. При селекции аптамеров к тканям рака молочной железы на 3-ем раунде селекции ДНК-аптамеры стабильно пропадали (на геле исчезали характерные для них полосы) и селекцию приходилось начинать заново.…”