High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers

Abstract

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. ‘High-throughput sequencing fluorescent ligand interaction profiling’ (HiTS–FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS–FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS–FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

Massively Parallel Selection of NanoCluster Beacons

“…Our chip screening platform can facilitate the development of new chemical sensors based on DNA‐templated AgNCs [ 26 ] or the study of other metal nanoclusters templated in DNA. [ 27 ] The chemical repertoire of natural nucleic acids can be expanded by connecting functional moieties to the alkyne‐modified C5 site on dU, [ 28 ] thus allowing us to study the interactions between a wide variety of functional moieties and AgNCs on NGS chips. The discoveries of critical zones and interaction hotspots lead to the zipper‐bag model, which provides us with a picture on how surrounding nucleobases can interact with the AgNCs encapsulated in NCBs and control their chemical yields and emission properties.…”

Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics

“…The obtained knowledge about molecular mechanisms will allow better rational molecular design, for example, to develop efficient biocatalysts for enzymatic production processes (Hauer, 2020). Such design and also direct screening of variant interactions with molecular or cellular targets can be employed in molecular detection assays, diagnostics, and therapeutics, for example, by development of specific aptamers and antibodies (Buglak et al, 2020;Drees and Fischer, 2021;Mamet et al, 2019;Norman et al, 2020;Wu et al, 2022). The ability to detect low-affinity interactions may be highly relevant in developing new therapeutics.…”

“…In the past decade, binding assays on next-generation sequencing chips have been applied to study the influence of DNA, RNA, peptide, and protein sequence for numerous biological systems (Andreasson et al, 2022(Andreasson et al, , 2020Becker et al, 2019aBecker et al, , 2019bBonilla et al, 2021;Boyle et al, 2017;Buenrostro et al, 2014;Denny and Greenleaf, 2019;Denny et al, 2018;Drees and Fischer, 2021;Jarmoskaite et al, 2019;Jones et al, 2021;Jung et al, 2017;Layton et al, 2019;Li et al, 2020;Mamet et al, 2019;Nutiu et al, 2011;Ober-Reynolds et al, 2022;Ozer et al, 2015;Perkel, 2018;She et al, 2017;Svensen et al, 2016;Tome et al, 2014;Wu et al, 2019Yesselman et al, 2019). In the next decade, we expect to obtain a more detailed picture by transitioning from averaging over the roughly thousand molecules in a cluster to measurements of individual molecules.…”

The Influence of Protein Charge and Molecular Weight on the Affinity of Aptamers

“…However, common to all SELEX methods is that the selection itself remains a “black box”—largely random and incomprehensible. Recently introduced high-throughput selection methods are increasingly providing insight into the process [ 6 , 7 ]. In addition, the first systematic studies and database analyses were conducted regarding the influence of selection conditions on the selection process [ 8 ].…”