Highly Parallel Single-Molecule Amplification Approach Based on Agarose Droplet Polymerase Chain Reaction for Efficient and Cost-Effective Aptamer Selection

Abstract

We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.

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

“…In another study, aptamers within a pre-enriched library were singly contained in agarose droplets generated by a microfluidic system, clonally amplified, and later tested individually for target binding using flow cytometry. 37 The agarose droplet-based approach led to isolation of an ssDNA aptamer with 25 nmol/l affinity against Shp2 protein, a protein tyrosine phosphatase implicated in cancer. However, starting library size restrictions, availability of a FACS instrument and fabrication of microfluidic devices represent major hurdles for wide use of Particle Display- and agarose droplet-based approaches.…”

“…38,39 The two methods would be most beneficial when used as an alternative method of selection following one or a few rounds of selection with other methods, as has been done. 36,37 …”

The Clinical Application of Aptamers: Future Challenges and Prospects

“…To reduce and simplify this timeconsuming, labor-intensive process, agarose droplet SELEX (Fig. 14.3) and surface display SELEX were developed for efficiently screening aptamers from a complex ssDNA library by employing single-molecule emulsion PCR (ePCR) in agarose droplets [39] or on the surface of microbeads [40]. Compared to the conventional cloning-sequencing-synthesis-screening work flow, these methods take advantage of the compartmentalization of droplets and allow rapid screening of individual DNA sequences from an enriched library prior to knowing their exact sequence information, thereby accelerating and simplifying the entire process at reduced cost.…”

Recent Progress in Aptamer‐Based Functional Probes for Bioanalysis and Biomedicine

“…In our group, we have developed asingle-molecule emulsion, PCR-assisted, aptamer screening method [59] to simplifyt he screening process.I nt he design,apre-enriched DNA library against SH2 domain-containing phosphatase(Shp2) was diluted and encapsulatedi nto individual uniform agarose droplets at as ingle molecule per dropletc oncentrationu sing af low-focusing method. [60] After the dropletP CR amplification,o nly amplified DNA droplets were picked out and incubated with target to determine the binding affinity.O nly those sequences with strong bindinga ffinity were chosen as aptamers for sequencing to identify the exact sequence directly used for biomedical applicationsw ithout knowing the exact sequence information. Compared to the traditional cloning-sequencing-synthesisscreening workflow,t his methoda voidst edious cloning,l argescale sequence and DNA synthesis and is thus more rapid, efficient, and cost effective.…”