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  • Primer
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In vitro selection of aptamers and their applications

Nature Reviews Methods Primers volume 3, Article number: 54 (2023) Cite this article

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Abstract

The introduction of the in vitro evolution method known as SELEX (systematic evolution of ligands by exponential enrichment) more than 30 years ago led to the conception of versatile synthetic receptors known as aptamers. Offering many benefits such as low cost, high stability and flexibility, aptamers have sparked innovation in molecular diagnostics, enabled advances in synthetic biology and facilitated new therapeutic approaches. The SELEX method itself is inherently adaptable and offers near limitless possibilities in yielding functional nucleic acid ligands. This Primer serves to provide guidance on experimental design and data analysis, and highlights new growth areas for this impactful technology.

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Fig. 1: General outline of the SELEX process and aptamer identification.
Fig. 2: Decision-making for SELEX design.
Fig. 3: Representative analysis of the in vitro selection process.
Fig. 4: Representative characterization techniques of aptamer candidates.
Fig. 5: Aptamers are powerful tools for molecular imaging.
Fig. 6: Aptamers that bind to light up fluorophores.

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Acknowledgements

The authors thank the reviewers for their valuable feedback in the preparation of this article. M.C.D. and M.M. thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for Discovery Grant funding. M.M. also thanks the CRC programme for her Tier II Research Chair. P.M. acknowledges grant no. R35 GM139336 from the National Institute of General Medical Sciences (NIGMS).

Author information

Author notes

  1. These authors contributed equally: Maria C. DeRosa, Amy Lin, Prabodhika Mallikaratchy, Erin M. McConnell, Maureen McKeague, Rutika Patel, Sarah Shigdar.

Authors and Affiliations

  1. Department of Chemistry and Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada

    Maria C. DeRosa & Erin M. McConnell

  2. Department of Chemistry, Faculty of Science, McGill University, Montreal, Québec, Canada

    Amy Lin & Maureen McKeague

  3. Department of Molecular, Cellular, and Biomedical Sciences, City University of New York School of Medicine, New York, NY, USA

    Prabodhika Mallikaratchy

  4. Department of Chemistry and Biochemistry, CUNY Graduate Center, New York, NY, USA

    Prabodhika Mallikaratchy & Rutika Patel

  5. Department of Molecular, Cellular and Developmental Biology, CUNY Graduate Center, New York NY, NY, USA

    Prabodhika Mallikaratchy

  6. Department of Pharmacology and Therapeutics, Faculty of Medicine and Health Sciences, McGill University, Montreal, Québec, Canada

    Maureen McKeague

  7. School of Medicine, Deakin University, Geelong, Victoria, Australia

    Sarah Shigdar

  8. Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Deakin University, Geelong, Victoria, Australia

    Sarah Shigdar

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Related links

APTANI: http://aptani.unimore.it/downloads.php

AptaSUITE: https://drivenbyentropy.github.io/

AptCompare: https://bitbucket.org/shiehk/aptcompare/src/master/

BLAST: Basic Local Alignment Search Tool: https://blast.ncbi.nlm.nih.gov/Blast.cgi

FASTAptamer: https://burkelab.missouri.edu/fastaptamer.html

Galaxy: https://usegalaxy.org/

MEMERIS: https://cs.stanford.edu/people/hillerm/Data/MEMERIS/

MEME Suite: https://meme-suite.org/meme/doc/download.html

MPBind: https://morgridge.org/research/regenerative-biology/software-resources/mpbind/

National Library of Medicine: https://www.ncbi.nlm.nih.gov/sra

PPAI: http://39.96.85.9/PPAI/

Protein Data Bank: https://www.rcsb.org/search

RaptRanker: https://github.com/hmdlab/RaptRanker

US National Library of Medicine’s online database: https://clinicaltrials.gov/

Supplementary information

Glossary

Counter-selections

Incubation of the library with a similar molecule(s) allows for increased stringency as sequences that have cross-reactivity to the similar molecule(s) are removed from the selection. Typically, counter-selections would occur after a negative selection and before a positive selection.

Enrichment

A measure of the progress of a selection, library enrichment describes the degree of convergence of the library on sequences with the desired properties.

Heterogeneous library

The pool of oligonucleotide sequences from which aptamers are selected, typically consisting of a random nucleotide core of between 20 and 60 nucleotides, flanked by fixed regions used for library amplification. In a typical systematic evolution of ligands by exponential enrichment (SELEX) experiment, the library contains 101–1015 random sequences (1–10 nmol).

Levenshtein distance

An algorithm that measures the difference between two sequences. In sequence comparison, the number of substitutions, insertions or deletions will affect the value. A common example of where this is used is for the BLAST: Basic Local Alignment Search Tool maintained by the National Institutes of Health (NIH).

Negative selections

It is important to remove sequences from the library that may bind to the selection matrix (such as magnetic beads, agarose beads and nitrocellulose paper). This is achieved by incubating the library with only the selection matrix prior to the positive selection.

Partitioning

The process by which target binding oligonucleotide sequences are separated from non-binding sequences.

Partitioning method

One of multiple methods to achieve partitioning, including, for example, affinity chromatography, nitrocellulose binding and bead-based separation.

Positive selection

The incubation of the selection library with the desired target.

Sequence space

Sequence space describes all of the possible sequences that could exist with a library having a random region of length N. For example, a library with a random region 24 nucleotides long would have 424 different unique possibilities (2.8 × 1014 sequences).

Selectivity

The ability of an aptamer to differentiate between components in a mixture.

Specificity

The ultimate selectivity, the ability of an aptamer to interact with only a single component in a mixture.

Stringency

Selection pressure used during the systematic evolution of ligands by exponential enrichment (SELEX) experiment to increase the competition between strong and weak binders, and ultimately increase aptamer suitability (affinity and specificity). Stringency can depend on several factors, including the partitioning method, target concentration, buffer composition and counter-selection rounds.

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DeRosa, M.C., Lin, A., Mallikaratchy, P. et al. In vitro selection of aptamers and their applications. Nat Rev Methods Primers 3, 54 (2023). https://doi.org/10.1038/s43586-023-00238-7

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