Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening

Hunt, Eric A.; Tamanaha, Esta; Bonanno, Kevin; Cantor, Eric J.; Tanner, Nathan A.

doi:10.3389/fmolb.2021.670940

Cited by 7 publications

(4 citation statements)

References 25 publications

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“…This result may be due to the different targets and reaction conditions such as the concentration of divalent ion and the temperature both being different. In addition, although Ago proteins do not need the PAM (protospacer adjacent motif) sequence, there were still differences in the efficiency between different targets (Hunt et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

Tang

Wang²,

Wang³

et al. 2023

Front. Bioeng. Biotechnol.

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In spite of the development of genome-editing tools using CRISPR–Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5′-phosphorylated (5′-P) or 5′- hydroxylated (5′-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn2+ or Mg2+ and displayed no obvious preference for the 5′-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

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Section: Resultsmentioning

confidence: 99%

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

Tang

Wang²,

Wang³

et al. 2023

Front. Bioeng. Biotechnol.

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“…CE is an opticsbased system where a dye must be present on the RNA/DNA for detection. This methodology has shown potential in a variety of applications [61,[70][71][72][73][74][75][76][77][78][79]. The 3730 DNA Analyzer from ThermoFisher Scientific (Waltham, MA) was used in this study and processes 96 samples simultaneously.…”

Section: Overview Of Hiker Platformmentioning

confidence: 99%

High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation

Carter,

O’Brien,

Lund

et al. 2024

Preprint

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T7 RNA Polymerase (RNAP) is a well-studied and widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as a key analysis tool for probing the kinetic mechanism of T7 RNAP nucleotide addition. However, sequencing gels are both slow and low throughput limiting their utility for comprehensive enzyme analysis. Here, we report the development of HiKER; (High-throughputKinetics using CapillaryElectrophoresis andRobotics) a high-throughput pipeline to quantitatively measure enzyme kinetics. We adapted a traditional polymerase misincorporation assay for fluorescent detection at scale allowing rapid estimates of RNAP misincorporation in different experimental conditions. In addition, high-throughput kinetics reactions were automated using an open-source OT-2 liquid handling robot. The platform allows multiple weeks’ worth of data to be collected in mere days. Using this platform, ∼1500 time points were collected in a single workday. T7 RNAP exhibited dramatic differences in both observed rate constant and amplitude depending on the mismatch examined. An average misincorporation frequency of ∼45 misincorporations per million bases was estimated using HiKER and is consistent with previous observations from next generation sequencing studies. Misincorporation time courses for T3 RNAP and Sp6 RNAP were similar to T7 RNAP suggesting conserved kinetic mechanisms. Interestingly, dramatic changes in the extent of misincorporation were observed in the three RNAPs depending on the mismatch. Extension from base mismatch experiments showed differences between T7, T3, and Sp6 RNAP. Sp6 RNAP was the slowest to extend from a mismatch followed by T7 RNAP and then T3 RNAP. Taken together the results presented here demonstrate the capabilities of HiKER to carry out high-throughput enzymology studies. Importantly, this pipeline and the corresponding analysis strategies are affordable, open-source, and broadly applicable to many enzymes.

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“…In a large-scale study using the guide panel for the TtAgo, patterns of preference for guide sequences were determined. Based on the results obtained in the course of this work, it is possible to optimize the using of TtAgo as a programmable endonuclease [ 143 , 186 ].…”

Section: Prospects For the Practical Application Of Prokaryotic Argon...mentioning

confidence: 99%

Prokaryotic Argonaute Proteins as a Tool for Biotechnology

et al. 2022

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Programmable nucleases are the most important tool for manipulating the genes and genomes of both prokaryotes and eukaryotes. Since the end of the 20th century, many approaches were developed for specific modification of the genome. The review briefly considers the advantages and disadvantages of the main genetic editors known to date. The main attention is paid to programmable nucleases from the family of prokaryotic Argonaute proteins. Argonaute proteins can recognize and cleave DNA sequences using small complementary guide molecules and play an important role in protecting prokaryotic cells from invading DNA. Argonaute proteins have already found applications in biotechnology for targeted cleavage and detection of nucleic acids and can potentially be used for genome editing.

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Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening

Cited by 7 publications

References 25 publications

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation

Prokaryotic Argonaute Proteins as a Tool for Biotechnology

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