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.
Highlights
Aptamers (latin aptus—suitable; greek μέρoς—part) are synthetically produced, single-stranded nucleic acid oligomers (e.g., DNA or RNA) or peptides which fold into unique 3D structures and thereby can bind target molecules [1,2]
While achieving similar affinity and specificity compared to antibodies, especially nucleic acid-based aptamers offer several advantages: (i) unlike antibodies produced in vivo, aptamers are synthesized entirely in vitro, an aspect desirable both for animal welfare reasons and for ensuring much greater batch stability; (ii) they are characterised by thermal stability, as well as (iii) the possibility to be adapted to a wide variety of environmental conditions; (iv) aptamers can be dried and reconstituted, which prolongs shelf life and facilitates application; (v) once developed and characterised, aptamers can be produced in large quantities and at low cost; (vi) targeted modification, e.g., for programmable pharmacokinetics, is possible
When selecting aptamers that are covalently bound to the flow cell via high-throughput sequencing (HiTS)–fluorescent ligand interaction profiling’ (FLIP), the fixed regions that are not bound to the flow cell can be cleaved off during the experiment to prevent potential steric hindrance within the FLIP, as well as interactions of these sequences with the variable region
Summary
Aptamers (latin aptus—suitable; greek μέρoς (méros)—part) are synthetically produced, single-stranded nucleic acid oligomers (e.g., DNA or RNA) or peptides which fold into unique 3D structures and thereby can bind target molecules [1,2]. HiTS can be performed after each round of selection and enables a more comprehensive analysis of the obtained aptamer pools This additional step allows the identification of functional and rare motifs, as well as the quantification of their abundance. The aptamer sequences to be investigated have to be identified before the microarray is synthesised; the pre-selection and sequencing of the obtained aptamer pool is necessary These two approaches were combined on a platform within an experiment called ‘high-throughput sequencing–fluorescent ligand interaction profiling’ (HiTS–FLIP). Several million DNA sequences can be analysed in parallel and without bias for their affinity and specificity towards a fluorescent target at equilibrium [28] This offers enormous potential for interaction studies.
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