reMelting curve analysis as a tool for enrichment monitoring in the SELEX process

Vanbrabant, Jeroen; Leirs, Karen; Vanschoenbeek, Katrijn; Lammertyn, Jeroen; Michiels, Luc

doi:10.1039/c3an01884a

Cited by 32 publications

(36 citation statements)

References 20 publications

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“…33 Although important, the above-mentioned methods extrapolate the selection for target-specific oligonucleotides from indirect events like library complexity or increased elution, which could be severely impacted by unforeseen biases common in SELEX protocols. The presented direct evaluation of the SELEX progression via SPR-based measurement of the target- affinity of the cycle-libraries overcomes this problem.…”

Section: ■ Discussionmentioning

confidence: 99%

More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

2015

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To address limitations in the production of DNA aptamers against small molecules, we introduce a DNA-based capture-SELEX (systematic evolution of ligands by exponential enrichment) protocol with long and continuous randomized library for more flexibility, coupled with in-stream direct-specificity monitoring via SPR and high throughput sequencing (HTS). Applying this capture-SELEX on tobramycin shows that target-specificity arises at cycle number 8, which is confirmed by sequence convergence in HTS analysis. Interestingly, HTS also shows that the most enriched sequences are already visible after only two capture-SELEX cycles. The best aptamers displayed K D of approximately 200 nM, similar to RNA and DNA-based aptamers previously selected for tobramycin. The lowest concentration of tobramycin detected on label-free SPR experiments with the selected aptamers is 20-fold smaller than the clinical range limit, demonstrating suitability for smalldrug biosensing.

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Section: ■ Discussionmentioning

confidence: 99%

More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

2015

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“…The 80‐mer library was combinatorially synthesized by mixing the DNA nucleotides A:C:G:T at a molar ratio of 3:3:2:2.4 to achieve equal probability incorporation of each nucleotide in the core region (Pollard et al, ). The 20‐mer flanking sequences and associated primers (Table S‐I; Supporting Information) are ones commonly employed in conventional SELEX (Janssen et al, ; Ouellet et al, ; Vanbrabant et al, ), and were utilized here due to their suitability for SYBR green qPCR amplification (Janssen et al, ). For this work, both flanking sequences had their reverse complement synthesized, with the complement to the 5′ and 3′ flanking sequences denoted in Table S‐I (Supporting Information) as 5′‐Comp and 3′‐Comp, respectively.…”

Section: Methodsmentioning

confidence: 99%

Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform

Ouellet

Foley

Conway

et al. 2015

Biotech & Bioengineering

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Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified.

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“…, by FACS, 68 EMSA, 47 filter binding, 127 or capillary electrophoresis (CE)), 128 or (iii) convergence of the aptamer species ( e.g. , by Restriction Fragment Length Polymorphism (RFLP), 129 melting curve, 130 Cot analysis, 131 or HPLC). 132 A recent study compared RFLP, melting curve, fluorescence, QPCR, and bulk binding affinity measurement assays to monitor the progress of SELEX against streptavidin concluded that these assays were all equally informative.…”

Section: Emerging Concepts In Selex and Aptamer Fieldsmentioning

confidence: 99%

New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization

Ozer

Pagano

Lis

2014

Molecular Therapy - Nucleic Acids

154

133

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Single-stranded oligonucleotide aptamers have attracted great attention in the past decade because of their diagnostic and therapeutic potential. These versatile, high affinity and specificity reagents are selected by an iterative in vitro process called SELEX, Systematic Evolution of Ligands by Exponential Enrichment. Numerous SELEX methods have been developed for aptamer selections; some that are simple and straightforward, and some that are specialized and complicated. The method of SELEX is crucial for selection of an aptamer with desired properties; however, success also depends on the starting aptamer library, the target molecule, aptamer enrichment monitoring assays, and finally, the analysis and characterization of selected aptamers. Here, we summarize key recent developments in aptamer selection methods, as well as other aspects of aptamer selection that have significant impact on the outcome. We discuss potential pitfalls and limitations in the selection process with an eye to aid researchers in the choice of a proper SELEX strategy, and we highlight areas where further developments and improvements are desired. We believe carefully designed multiplexed selection methods, when complemented with high-throughput downstream analysis and characterization assays, will yield numerous high-affinity aptamers to protein and small molecule targets, and thereby generate a vast array of reagents for probing basic biological mechanisms and implementing new diagnostic and therapeutic applications in the near future.

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reMelting curve analysis as a tool for enrichment monitoring in the SELEX process

Cited by 32 publications

References 20 publications

More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

More DNA–Aptamers for Small Drugs: A Capture–SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing

Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform

New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization

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