Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods

Abstract

The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA "parts" in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid-or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.

Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

“…An AuNP-RCA biosensor had been used for pathogen detection ( Shi et al, 2014 ), which could identify 0.5 pM of synthetic oligonucleotides or 0.5 ng/ml of genomic DNA. Even so, RCA only could detect the circular molecules of DNA or double-stranded DNA ( Hadi et al, 2020 ), which limited its application. Factors affecting the assay performance is discussed in the following section:…”

“…Immunoassays are the most popular assays for molecular diagnostics ( Cox et al, 2019 ; Maggio, 2018 ; Poschenrieder et al, 2019 ; Organization (W.H, 2020) . Various state-of-the-art technologies were combined with the traditional immunoassay and applied in clinical diagnostics, such as a single-molecule array (Simoa) ( Mathian et al, 2019 ; Cohen et al, 2020 ) and rolling circle amplification (RCA) ( Bhat and Rao, 2020 ; Hadi et al, 2020 ). Among them, enzyme-linked immunosorbent assay (ELISA) is the gold standard of in vitro diagnostics to analyze biomarkers and important analytes in healthcare and diversified analytical settings ( Tabatabaei et al, 2020 ).…”

Amplified DNA Heterogeneity Assessment with Oxford Nanopore Sequencing Applied to Cell Free Expression Templates

“…Fragments of incompletely synthesized DNA may contribute in this step, forming errored circular templates. Therefore, pre-amplifying with high-fidelity enzymes, such as Q5, as opposed to OneTaq [ 6 ], is considered the optimal strategy for RCA compared to directly making circular from the vendor DNA [ 36 ].…”

HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale

“…Moreover, the LCR as the first step of the workflow directly enables DNA recombination and circularization to create its own focused DNA libraries and RCA templates, whereas other systems depend on presynthesized DNA libraries for starting. Finally, starting with a DNA-part library, HyperXpress can be executed in 10 h compared to at least 12 h (Kumar and Chernaya, 2009;Dopp et al, 2019a;Dopp et al, 2019b;Hadi et al, 2020).…”

“…But the potential of these RCA-CFPS systems has not yet been fully exhausted, as their practical implementation is still accompanied by cumbersome work steps and increased material consumption (Dopp et al, 2019b). Thus previous RCA-CFPS systems suffer from high total reaction volumes of 15 μl, long execution times of at least 12 h, a high number of workflow steps of six or more and changing of reaction vessels (Dopp et al, 2019b;Hadi et al, 2020).…”