Synthetic biological approaches for RNA labelling and imaging: design principles and future opportunities

Pauff, Steven M.; Withers, Jamie M.; McKean, Iain J. W.; Mackay, Simon P.; Burley, Glenn A.

doi:10.1016/j.copbio.2017.04.003

Cited by 9 publications

(9 citation statements)

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“…Although generally dominated by chemical synthesis, the development of engineered DNA polymerases for the synthesis of modified nucleic acids is a growing field, and enzymatic modification of NTPs and RNA oligonucleotides answers the need for unnatural oligonucleotides in synthetic biology, imaging, and therapeutics. A detailed description of the available modification methods is beyond the scope of this review; however, we direct the interested reader to a series of reviews that highlight the latest advancements in polymerase engineering and enzymatic synthesis of modified RNAs [ 72 – 76 ].…”

Section: Sample Preparationmentioning

confidence: 99%

A guide to large-scale RNA sample preparation

et al. 2018

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RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods. Graphical abstractIn this review we present different methods for large-scale production and purification of RNAs for structural and biophysical studies

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Section: Sample Preparationmentioning

confidence: 99%

A guide to large-scale RNA sample preparation

et al. 2018

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“…An engineered Thg1 system that can be directed towards a variety of substrates would thus be highly useful and add to the current toolbox of RNA and DNA manipulating enzymes. 29 We are currently engineering Figure 6. Mutational analysis of Thg1 enzymes.…”

Section: P Aerophilum Thg1 Repairs Truncated Trna Substratesmentioning

confidence: 99%

Minimal requirements for reverse polymerization and tRNA repair by tRNA^His guanylyltransferase

et al. 2017

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tRNA guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNA maturation. These enzymes normally catalyze a single 5' guanylation of tRNA lacking the essential G identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNA substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

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“…However, the use of fluorescence-labelled DNA or RNA aptamers is an expensive option for the characterization of large populations. Apart from that, colorimetric readout is easily adaptable to DNA aptamers (through biotin or digoxigenin labelling), but it is less optimized for RNA aptamers (because of the inherent technical difficulties associated to RNA labelling) [20,21,22]. Furthermore, colorimetric reactions generally show a limited sensitivity and require additional, time-consuming recognition reactions involving either streptavidin or an anti-digoxigenin antibody labelled with horseradish peroxidase (HRP) or alkaline phosphatase (AP) [23].…”

Section: Introductionmentioning

confidence: 99%

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

et al. 2019

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Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

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Synthetic biological approaches for RNA labelling and imaging: design principles and future opportunities

Cited by 9 publications

References 50 publications

A guide to large-scale RNA sample preparation

A guide to large-scale RNA sample preparation

Minimal requirements for reverse polymerization and tRNA repair by tRNA^His guanylyltransferase

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

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Synthetic biological approaches for RNA labelling and imaging: design principles and future opportunities

Cited by 9 publications

References 50 publications

A guide to large-scale RNA sample preparation

A guide to large-scale RNA sample preparation

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

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Product

Resources

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Minimal requirements for reverse polymerization and tRNA repair by tRNA^His guanylyltransferase