Optimized quantitative PCR analysis of random DNA aptamer libraries

Pearson, Keenan; Doherty, Caroline; Zhang, Drason; Becker, Nicole A.; Maher, Jim

doi:10.1016/j.ab.2022.114712

Cited by 5 publications

(4 citation statements)

References 16 publications

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“…Progress at each round was monitored by qPCR, including a BT-free mock selection at round 9 and an APEX2-free mock selection at round 15 to demonstrate that library enrichment depended on the presence of each reagent (Fig. S2B–D ) ( 20 ). qPCR results indicated recovery above background at each round, though exponential enrichment was not achieved over these cycles.…”

Section: Resultsmentioning

confidence: 99%

Peroxidase proximity selection to identify aptamers targeting a subcellular location

et al. 2023

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The efficient and specific delivery of functional cargos such as small molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3-dimensional structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here we describe Peroxidase Proximity Selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of a six-fold larger IgG antibody.

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Section: Resultsmentioning

confidence: 99%

Peroxidase proximity selection to identify aptamers targeting a subcellular location

et al. 2023

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“…The progress of each selection was monitored by subjecting recovered aptamer libraries to qPCR. 26 In general, library recovery increased across selection rounds (Fig. S1).…”

Section: Aptamer Selectionmentioning

confidence: 95%

“…Samples were transferred to a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad Laboratories, 1854095) and subjected to thermal cycling using a protocol of 95˚C, 30s; 40×(95˚C, 15s; 51˚C, 30s; 72˚C, 30s), and interrogating SYBR Green fluorescence after the anneal step of each cycle as previously described. 26 Raw fluorescence data were then analyzed using CFX Maestro™ software.…”

Section: Monitoring Library Recoverymentioning

confidence: 99%

DNA aptamers that modulate biological activity of model neurons

Rolli,

Pearson,

Wilbanks

et al. 2024

Preprint

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There is an urgent need for agents that promote health and regeneration of cells and tissues, specifically to treat diseases of the aging nervous system. Age-associated nervous system degeneration and various diseases are driven by many different biochemical stresses, often making it difficult to target any one disease cause. Our laboratory has previously identified DNA aptamers with apparent regenerative properties in murine models of multiple sclerosis by selecting aptamers that bind oligodendrocyte membrane preparations. Here, we screened vast libraries of molecules (∼1014unique DNAs) for the ability to bind cultured human SH-SY5Y neuroblastoma cells as model neurons to demonstrate the feasibility of identifying biologically active aptamers by cycles of cell selection. Many of these DNA aptamers bind undifferentiated and differentiated cultured SH-SY5Y cells. Several of these aptamers modulate the biological activity of SH-SY5Y cells upon treatment in culture.

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“…However, as many high-a nity aptamer candidates as possible should be isolated and without insight into the course of the process, it is di cult to determine the right time to stop the selection, hence it is essential to monitor the progress of selection using fast and simple, but at the same time precise and reliable techniques to signi cantly increase the chances of a nally successful and fast selection. 22,25 In turn the complexity of the scienti c task to perform a fast, reliable and nally successful e cient SELEX may be discouraging and the reason for the undeservedly slow development of the eld in the rst 25 years of aptamer research with a remarkable acceleration in the last few years in parallel with the invention of monitoring techniques for the SELEX success.…”

Section: Introductionmentioning

confidence: 99%

IMPATIENT-qPCR: melting-temperature-shift based evolution monitoring of successful aptamer development in SELEX processes

Kissmann,

Bolotnikov,

et al. 2023

Preprint

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SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting-temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC-content and Tm. Based on nine successful previous processes we show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general.

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Optimized quantitative PCR analysis of random DNA aptamer libraries

Cited by 5 publications

References 16 publications

Peroxidase proximity selection to identify aptamers targeting a subcellular location

Peroxidase proximity selection to identify aptamers targeting a subcellular location

DNA aptamers that modulate biological activity of model neurons

IMPATIENT-qPCR: melting-temperature-shift based evolution monitoring of successful aptamer development in SELEX processes

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