Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

Kupakuwana, Gillian V.; Crill, James E.; McPike, Mark P.; Borer, Philip N.

doi:10.1371/journal.pone.0019395

Cited by 57 publications

(50 citation statements)

References 43 publications

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“…This result shows that copy number from a sequenced pool and affinity for the target may only be associated under certain conditions 24,34 , in contrast with a prior report implying a direct correlation 35 . It also highlights the benefit of monitoring enrichment by NGS, rather than solely by the % eluted strategy common in most selections, which may vary naturally from one round to the next as higher stringency conditions are applied.…”

Section: -3 Ggaatggatccacatccatgggagggttggaagggaggggcccggggtgggccatcontrasting

confidence: 96%

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Martin

Smith

Warren

et al. 2015

JoVE

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Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

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Section: -3 Ggaatggatccacatccatgggagggttggaagggaggggcccggggtgggccatcontrasting

confidence: 96%

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Martin

Smith

Warren

et al. 2015

JoVE

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“…Due to this complexity, interpreting ligated P2 sequences enriched or depleted from the initial P2-5N library as being more or less fit for ligation requires validating data to avoid misleading conclusions (Ditzler et al 2013;Kupakuwana et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

et al. 2016

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The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

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“…(8) published studies that utilized high-throughput sequencing to shorten the initial aptamer selection time. However, while both groups were able to identify aptamers that bind to the intended target, they either relied on multiple rounds of selection and sequencing (7) or limited the flexibility available in the sequence space by only analyzing fixed-length sequences (8). …”

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confidence: 99%

Aptamer Selection by High-Throughput Sequencing and Informatic Analysis

et al. 2011

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Traditional methods for selecting aptamers require multiple rounds of selection and optimization in order to identify aptamers that bind with high affinity to their targets. Here we describe an assay that requires only one round of positive selection followed by high-throughput DNA sequencing and informatic analysis in order to select high-affinity aptamers. The assay is flexible, requires less hands on time, and can be used by laboratories with minimal expertise in aptamer biology to quickly select high-affinity aptamers to a target of interest. This assay has been utilized to successfully identify aptamers that bind to thrombin with dissociation constants in the nanomolar range.

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Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

Cited by 57 publications

References 43 publications

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

Aptamer Selection by High-Throughput Sequencing and Informatic Analysis

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