Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

Stuart, Christopher H; Riley, Kathryn R.; Boyacioglu, Olcay; Herpai, Denise; Debinski, Waldemar; Qasem, Shadi; Marini, Frank C.; Colyer, Christa L.; Gmeiner, William H.

doi:10.1038/mtna.2016.91

Cited by 34 publications

(26 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The aptamer content could be increased from 0.4% in the original library before selection to >15% in the CE-selected fraction. A similar strategy was applied for the de novo selection of aptamers for vitronectin [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Stoltenburg

Strehlitz

2018

IJMS

View full text Add to dashboard Cite

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

show abstract

Section: Resultsmentioning

confidence: 99%

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Stoltenburg

Strehlitz

2018

IJMS

View full text Add to dashboard Cite

show abstract

“…These are not the unique literature examples of aptamer optimization obtained by dimerization of known aptamer sequences. Indeed, in the last decade a number of works described improved aptamers realized by engineering bivalent or multivalent analogues of the selected oligonucleotide, not only for VEGF, but also for thrombin [19,[22][23][24][25][26][27], mIgM (B-cell receptor) [28,29], and other biologically relevant targets [30][31][32], so that this strategy can be considered among those of choice to increase the overall efficacy of selected aptamers [33][34][35].…”

Section: Introductionmentioning

confidence: 99%

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Riccardi

Musumeci

Platella

et al. 2020

IJMS

View full text Add to dashboard Cite

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.

show abstract

“…Therefore, the in vitro DNA and RNA selection processes performed exemplify the need for an experimental approach that allows the screening of the binding affinity and specificity of the selected aptamers, beyond their individual sequences or secondary structures. Assays that effectively quantify aptamer-target interactions include colorimetric ELONA [23], flow cytrometry [36], surface plasmon resonance (SPR) [37], isothermal titration calorimetry [38], EMSA [39], or capillary electrophoresis [40]. However, each assay shows some benefits and drawbacks, its suitability depending on both the target features and the type of aptamers (i.e., DNA or RNA) selected [13,14,15].…”

Section: Discussionmentioning

confidence: 99%

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

et al. 2019

View full text Add to dashboard Cite

Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

show abstract

Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

Cited by 34 publications

References 41 publications

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

Contact Info

Product

Resources

About