High-Throughput (HT) SELEX combines SELEX (Systematic Evolution of Ligands by EXponential Enrichment), a method for aptamer discovery, with massively parallel sequencing technologies. This emerging technology provides data for a global analysis of the selection process and for simultaneous discovery of a large number of candidates but currently lacks dedicated computational approaches for their analysis. To close this gap, we developed novel in-silico methods to analyze HT-SELEX data and utilized them to study the emergence of polymerase errors during HT-SELEX. Rather than considering these errors as a nuisance, we demonstrated their utility for guiding aptamer discovery. Our approach builds on two main advancements in aptamer analysis: AptaMut—a novel technique allowing for the identification of polymerase errors conferring an improved binding affinity relative to the ‘parent’ sequence and AptaCluster—an aptamer clustering algorithm which is to our best knowledge, the only currently available tool capable of efficiently clustering entire aptamer pools. We applied these methods to an HT-SELEX experiment developing aptamers against Interleukin 10 receptor alpha chain (IL-10RA) and experimentally confirmed our predictions thus validating our computational methods.
Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is a well established experimental procedure to identify aptamers - synthetic single-stranded (ribo)nucleic molecules that bind to a given molecular target. Recently, new sequencing technologies have revolutionized the SELEX protocol by allowing for deep sequencing of the selection pools after each cycle. The emergence of High Throughput SELEX (HT-SELEX) has opened the field to new computational opportunities and challenges that are yet to be addressed. To aid the analysis of the results of HT-SELEX and to advance the understanding of the selection process itself, we developed AptaCluster. This algorithm allows for an efficient clustering of whole HT-SELEX aptamer pools; a task that could not be accomplished with traditional clustering algorithms due to the enormous size of such datasets. We performed HT-SELEX with Interleukin 10 receptor alpha chain (IL-10RA) as the target molecule and used AptaCluster to analyze the resulting sequences. AptaCluster allowed for the first survey of the relationships between sequences in different selection rounds and revealed previously not appreciated properties of the SELEX protocol. As the first tool of this kind, AptaCluster enables novel ways to analyze and to optimize the HT-SELEX procedure. Our AptaCluster algorithm is available as a very fast multiprocessor implementation upon request.
To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.
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.
Highly-constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly-constrained peptides are challenging to prepare. Here we present a method which utilizes two robust, orthogonal chemical steps to create highly-constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.
SUMMARY
Aptamers, short RNA or DNA molecules that bind distinct targets with high affinity and specificity, can be identified using High Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT-SELEX). But scalable analytic tools for understanding sequence-function relationships from diverse HT-SELEX data are not available. Here, we present AptaTRACE, a computational approach that leverages the experimental design of the HT-SELEX protocol, RNA secondary structure, and the potential presence of many secondary motifs to identify sequence-structure motifs that show a signature of selection. We apply AptaTRACE to identify nine motifs in C-C chemokine receptor type 7 targeted by aptamers in an in vitro cell-SELEX experiment. We experimentally validate two aptamers whose binding required both sequence and structural features. AptaTRACE can identify low-abundance motifs, and we show through simulations that because of this it could lower HT-SELEX cost and time by reducing the number of selection cycles required. AptaTRACE is available for download at www.ncbi.nlm.nih.gov/CBBresearch/Przytycka/index.cgi#aptatools.
Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.
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