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2015 ; 43
(21
): e139
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Characterisation of aptamer-target interactions by branched selection and
high-throughput sequencing of SELEX pools
#MMPMID26163061
Dupont DM
; Larsen N
; Jensen JK
; Andreasen PA
; Kjems J
Nucleic Acids Res
2015[Dec]; 43
(21
): e139
PMID26163061
show ga
Nucleic acid aptamer selection by systematic evolution of ligands by exponential
enrichment (SELEX) has shown great promise for use in the development of research
tools, therapeutics and diagnostics. Typically, aptamers are identified from
libraries containing up to 10(16) different RNA or DNA sequences by 5-10 rounds
of affinity selection towards a target of interest. Such library screenings can
result in complex pools of many target-binding aptamers. New high-throughput
sequencing techniques may potentially revolutionise aptamer selection by allowing
quantitative assessment of the dynamic changes in the pool composition during the
SELEX process and by facilitating large-scale post-SELEX characterisation. In the
present study, we demonstrate how high-throughput sequencing of SELEX pools,
before and after a single round of branched selection for binding to different
target variants, can provide detailed information about aptamer binding sites,
preferences for specific target conformations, and functional effects of the
aptamers. The procedure was applied on a diverse pool of
2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin
plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard
selection. The results demonstrate that it is possible to perform large-scale
detailed characterisation of aptamer sequences directly in the complex pools
obtained from library selection methods, thus without the need to produce
individual aptamers.