Reverse Transcriptases: From Discovery and Applications to Xenobiology

Huber, Luisa B; Betz, Karin; Marx, Andreas

doi:10.1002/cbic.202200521

Cited by 7 publications

(3 citation statements)

References 145 publications

(140 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This reaction can proceed either in a template-dependent or template-independent manner depending on the nature of the polymerase. Production of modified oligonucleotides is readily accessible by this approach provided the corresponding, chemically altered nucleoside triphosphates that are used in lieu of their natural counterparts are tolerated as substrates by the polymerases 23 . The potential of this method is showcased by the industrial production of mRNA vaccines which consist of long (>4000 nt), single-stranded RNA oligonucleotides containing modified nucleotides (mainly N 1-methyl-pseudouridine) 24 , 25 .…”

Section: Introductionmentioning

confidence: 99%

Controlled enzymatic synthesis of oligonucleotides

Pichon,

Hollenstein

2024

Commun Chem

View full text Add to dashboard Cite

Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.

show abstract

Section: Introductionmentioning

confidence: 99%

Controlled enzymatic synthesis of oligonucleotides

Pichon,

Hollenstein

2024

Commun Chem

View full text Add to dashboard Cite

show abstract

“…These and other potential benefits stimulate the ongoing efforts to create new high-temperature RTases engineered from Taq pol and other thermostable DNA polymerases. Successful examples of enhancement of the RTase activity in Taq pol via introduction of one or more point mutations have long been known [32] [33] [34] [35] [36] [37] [24] [38]. Of note, in different articles, modifications at completely different sites have had similar effects on this property.…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Reverse Transcriptase Function in Taq Polymerase via AI-driven Multiparametric Rational Design

Tomilova,

Russkikh,

et al. 2024

Preprint

View full text Add to dashboard Cite

Modification of natural enzymes to introduce new properties and enhance existing ones is a central challenge in bioengineering. This study is focused on the development of Taq polymerase mutants that show enhanced reverse transcriptase (RTase) activity while retaining other desirable properties such as fidelity, 5'-3' exonuclease activity, effective deoxyuracil incorporation, and tolerance to locked nucleic acid (LNA)-containing substrates. Our objective was to use AI-driven rational design combined with multiparametric wet-lab analysis to identify and validate Taq polymerase mutants with an optimal combination of these properties. The experimental procedure was conducted in several stages: 1) On the basis of a foundational paper, we selected 18 candidate mutations known to affect RTase activity across six sites. These candidates, along with the wild type, were assessed in the wet lab for multiple properties to establish an initial training dataset. 2) A ridge regression model was trained on this dataset to predict the enzymes properties. This model enabled us to select 14 new candidates for further experimental testing. 3) We refined our predictive model using Gaussian process regression and trained it on an expanded dataset now including 33 data points. 4) Leveraging the refined model, we screened in silico over 27 million potential mutations, thus selecting 16 for detailed wet-lab evaluation. Through this iterative data-driven approach, we identified 18 enzymes that not only manifested considerably enhanced RTase activity but also retained a balance of other required properties. These enhancements were generally accompanied by lower Kd, moderately reduced fidelity, and greater tolerance to noncanonical substrates, thereby illustrating a strong interdependence among these traits. Several enzymes validated via this procedure were effective in single-enzyme real-time reverse-transcription PCR setups, implying their utility for the development of new tools for real-time reverse-transcription PCR technologies, such as pathogen RNA detection and gene expression analysis. This study illustrates how AI can be effectively integrated with experimental bioengineering to enhance enzyme functionality systematically. Our approach offers a robust framework for designing enzyme mutants tailored to specific biotechnological applications. The results of our biological activity predictions for mutated Taq polymerases can be accessed at https://huggingface.co/datasets/nerusskikh/taqpol_insilico_dms.

show abstract

“…Canonical PEs contains two main components, a protein effector consisting of nCas9-H840A tethered with reverse transcriptase (RT) 1,2 at the C-terminus, and a prime editing guide RNA (pegRNA) containing a primer binding site (PBS) sequence and a RT template [3][4][5] . It can install desired insertions, deletions, and all 12 possible base-tobase conversions 3,[6][7][8] without double-strand breaks (DSBs) or donor DNA template, which enables PE to correct the majority of known human genetic disease-related mutations [8][9][10] .…”

Section: Introductionmentioning

confidence: 99%

SunTag-PE: a modular prime editing system enables versatile and efficient genome editing

Tian

Liu

et al. 2023

Preprint

View full text Add to dashboard Cite

Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full length PE effector size (6.3kb) was beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical transformation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag PE system that splits PE effectors into GCN4 nCas9 and single chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag PEs with one GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag PE strategy achieved editing levels comparable to canonical fused PE and higher than other split PE strategies (including sPE and MS2 PE) in both PE2 and PE3 forms with no increase in insertion/deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.

show abstract

Reverse Transcriptases: From Discovery and Applications to Xenobiology

Cited by 7 publications

References 145 publications

Controlled enzymatic synthesis of oligonucleotides

Controlled enzymatic synthesis of oligonucleotides

Enhancing the Reverse Transcriptase Function in Taq Polymerase via AI-driven Multiparametric Rational Design

SunTag-PE: a modular prime editing system enables versatile and efficient genome editing

Contact Info

Product

Resources

About