Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA

Ibach, Jenny; Dietrich, L.; Koopmans, Kyra R.M.; Nöbel, Nico; Skoupi, Marc; Brakmann, Susanne

doi:10.1016/j.jbiotec.2013.07.005

Cited by 42 publications

(35 citation statements)

References 34 publications

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“…This contrasts with the various R425 T7 RNA polymerase mutants developed by Brakmann and coworkers, which demonstrated 4-fold lower activities in the presence of modified nucleotides compared to natural nucleotides [19]. We found that 30 to 40 copies of fGmH RNA were synthesized by His-LAR T7 RNA polymerase in the presence of 2′-F-dGTP and 2′- O Me-dATP/dCTP/dUTP (data not shown).…”

Section: Resultscontrasting

confidence: 94%

“…Existing T7 RNA polymerase mutants do not efficiently generate 2′-fully modified RNAs, making direct selection of such libraries difficult [14, 15, 18, 19]. Work by Sousa and coworkers identified two T7 RNA polymerase mutants (Y639F and Sousa Variant Y639F/H784A) that allow for the efficient accommodation of 2′-F-pyrimidine ribonucleotides as substrates during in vitro transcription [20, 21].…”

Section: Introductionmentioning

confidence: 99%

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Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

2015

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When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2′ modification. This study aims to develop a novel class of highly stable, 2′-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2′-F-dG, 2′-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2′-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and further deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials.

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Section: Resultscontrasting

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

2015

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“…The most established method is the modification of the 2′-position of the ribose moieties in aptamers with fluoro and methoxy groups (18–20). Since these 2′-modified nucleotides can be introduced into RNA by transcription (21–23), 2′-modified RNA aptamers can be directly generated by SELEX (18,24,25). However, applying these 2′-modifications to DNA aptamers is often restricted, because of the different sugar conformation of the 2′-deoxyribose moieties in DNA from those of the 2′-modified ribose moieties, as well as the bulkiness of 2′-mothxy modifications (26).…”

Section: Introductionmentioning

confidence: 99%

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF₁₆₅toward pharmaceutical applications

2016

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A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165.

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“…Brakmann and colleagues have combined rational design with screening strategies to isolate a T7 mutant capable of efficient, fully substituted 2′-OMe-DNA synthesis [17].…”

Section: When Natural Is Not Enough: New Backbonesmentioning

confidence: 99%

Towards XNA nanotechnology: new materials from synthetic genetic polymers

Pinheiro¹,

Holliger

2014

Trends in Biotechnology

120

104

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HighlightsWe review recent advances in nucleic acid chemistry and polymerase engineering that have enabled the synthesis, replication, and evolution of a wide range of nucleic acid-like synthetic genetic polymers (XNAs) with improved chemical and biological stability.We discuss the likely biotechnological impact of the further development of XNA technology for the generation of novel ligands, enzymes, and nanostructures with tailor-made chemistry.

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Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA

Cited by 42 publications

References 34 publications

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF₁₆₅toward pharmaceutical applications

Towards XNA nanotechnology: new materials from synthetic genetic polymers

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Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2′-O-methyl-modified RNA

Cited by 42 publications

References 34 publications

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

Highly stable aptamers selected from a 2′-fully modified fGmH RNA library for targeting biomaterials

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165toward pharmaceutical applications

Towards XNA nanotechnology: new materials from synthetic genetic polymers

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Product

Resources

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Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF₁₆₅toward pharmaceutical applications