Elucidating the Determinants of Polymerase Specificity by Microfluidic-Based Deep Mutational Scanning

Nikoomanzar, Ali; Vallejo, Derek; Chaput, John C.

doi:10.1021/acssynbio.9b00104

Cited by 34 publications

(51 citation statements)

References 40 publications

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“…The Monarch DNA gel extraction kit was purchased from New England Biolabs (Ipswich, MA). Thermococcus gorgonarius (Tgo) DNA polymerase (exo-), Kod-RS ( 22 ) and Kod-RSGA ( 23 ) TNA polymerases (exo-), and Bst-LF were expressed and purified from E. coli as previously described ( 24 ). Sequenase™ Version 2.0 DNA polymerase was purchased from ThermoFisher (Pudong, Shanghai).…”

Section: Methodsmentioning

confidence: 99%

Transliteration of synthetic genetic enzymes

Wang

Liu

Shehabat

et al. 2021

Nucleic Acids Research

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Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.

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Section: Methodsmentioning

confidence: 99%

Transliteration of synthetic genetic enzymes

Wang

Liu

Shehabat

et al. 2021

Nucleic Acids Research

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“…In its first demonstration, DrOPS was used to identify a manganese-independent TNA polymerase from a site-saturation library of 8000 unique variants after a single round of high-throughput screening (Larsen et al ., 2016). More recently, DrOPS was combined with the protein-engineering approach of deep mutational scanning (Araya and Fowler, 2011), to map the sequence function relationships of a replicative DNA polymerase, Kod, isolated from the thermophilic archeae T. kodakarensis (Nikoomanzar et al ., 2019). The resulting enrichment profile provided an unbiased view of the ability of each single-point mutant to synthesize TNA.…”

Section: Engineering Polymerases By Directed Evolutionmentioning

confidence: 99%

Engineering polymerases for applications in synthetic biology

Nikoomanzar

Chim

Yik

et al. 2020

Quart. Rev. Biophys.

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DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

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“…FADS is also of highest sensitivity and could be as low as 2.5 nM of fluorescein at a 2-pl droplet (Hasan et al, 2019 ), which means less than a single turnover for all enzyme molecules from a single cell ( Table 1 ). Genotype is usually linked with fluorescent phenotype by fluorophore activation (Qiao et al, 2018 ), quencher release (Nikoomanzar et al, 2019 ), or coupled assay. In general, FADS is the primary choice if fluorescence could be achieved in the enzymatic reaction.…”

Section: Discussionmentioning

confidence: 99%

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Zhang

et al. 2021

Front. Chem.

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Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

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Elucidating the Determinants of Polymerase Specificity by Microfluidic-Based Deep Mutational Scanning

Cited by 34 publications

References 40 publications

Transliteration of synthetic genetic enzymes

Transliteration of synthetic genetic enzymes

Engineering polymerases for applications in synthetic biology

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

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