Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

Fraser, Lewis A.; Kinghorn, Andrew B.; Tang, Marco S. L.; Cheung, Yiu-ming; Lim, Bryce; Liang, Shuailong; Dirkzwager, Roderick M.; Tanner, Julian A.

doi:10.3390/molecules201219766

Cited by 19 publications

(17 citation statements)

References 89 publications

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“…[64][65][66][67][68] Through retroviral technology and fluorescence-activated cell sorting (FACS), a distinct cell population can be easily amplified based on the appropriate fluorescent phenotype. 69,70 Our goal is to ultimately obtain cell lines expressing two independent assays to provide a robust tool for the screening of antivirals. It is thus critical that we first prove these assays can be expressed independently and then tested in conjunction, whether by mixing cells or in the same cell background.…”

Section: Resultsmentioning

confidence: 99%

A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

et al. 2015

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Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread.

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Section: Resultsmentioning

confidence: 99%

A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

et al. 2015

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“…The aptamer was shown to stimulate extracellular matrix deposition relative to controls. Future work can improve binding affinity by microarray maturation, employ bioinformatics methods to modify the aptamer for greater functionality [ 48 ] or generate a library that could be used alongside a microfluidic selection method [ 49 ]. By such approaches, novel therapeutic aptamers can be further developed for a variety of human disease.…”

Section: Discussionmentioning

confidence: 99%

Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1

Tucker¹,

Kinghorn²,

Fraser³

et al. 2018

IJMS

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Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy.

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“…Our strategy expanded upon existing workflows to generate 'monoclonal' beads via on-bead ePCR with libraries of DNA templates. [6,14,16,22,32] On-bead ePCR has enabled high-throughput screening for interactions between transcription factors and DNA [16], aptamer selections, [15,22,33] detection and quantification of rare allelic mutations, [32] and high-throughput next-generation sequencing (NGS), [6,[34][35][36] among myriad applications. While these works successfully generated monoclonal beads and demonstrated downstream utility, none of them used a single particle strategy such as ours to assess ePCR in terms of the percentage of saturated beads and amplicon coverage per bead.…”

Section: Increasing Reverse Primer and Dntp Concentration Has No Effementioning

confidence: 99%

Optimization of On-Bead Emulsion Polymerase Chain Reaction Based on Single Particle Analysis

Siu

Liu

Chan

et al. 2020

Preprint

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Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring, the only methods that can emulsify libraries of ≥108 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study systematically quantifies the influence of tuning individual ePCR parameters by single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that increasing the polymerase concentration 20-fold in ePCR over recommended concentrations for conventional PCR was necessary to obtain sufficient PCR products. Surprisingly, dramatical increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.

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Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

Cited by 19 publications

References 89 publications

A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1

Optimization of On-Bead Emulsion Polymerase Chain Reaction Based on Single Particle Analysis

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