Studies of RNA Sequence and Structure Using Nanopores

Henley, Robert Y.; Carson, Spencer; Wanunu, Meni

doi:10.1016/bs.pmbts.2015.10.020

Cited by 27 publications

(22 citation statements)

References 59 publications

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“…Nanopore translocation is a powerful method to probe biopolymers as well as understanding how they interact with processive enzymes [8,15,26,[30][31][32][33][34][35]. Here we used it to clarify the structural determinants of xrRNAs resistance to exonuclease degradation.…”

Section: Discussionmentioning

confidence: 99%

Directional translocation resistance of Zika xrRNA

Suma

Coronel

Bussi

et al. 2020

Preprint

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xrRNAs from flaviviruses survive in host cells for their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, 5 and 3 , are driven through a pore. Pulling the 3 end, as done by replicases, elicits a progressive unfolding; pulling the 5 end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the 5 end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials.

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Section: Discussionmentioning

confidence: 99%

Directional translocation resistance of Zika xrRNA

Suma

Coronel

Bussi

et al. 2020

Preprint

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“…Moving beyond RT-based methods to direct sequencing of tRNAs and their modifications is another exciting development on the horizon. However, major advances must still be made in techniques such as nanopore sequencing because current technologies cannot be used to reliably sequence tRNA molecules in biological total RNA samples because they are likely unable to discriminate between more than two tRNAs at a time (Henley et al 2016;Poodari 2019). Additionally, efforts to characterize the resistance properties of alternative tRNA base modifications in nanopores are underway (Onanuga et al 2017) but still in their infancy.…”

Section: The Landscape Of Base Modifications In Plant Trnasmentioning

confidence: 99%

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

Warren

Salinas‐Giegé

Hummel

et al. 2019

Preprint

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Differences in tRNA expression have been implicated in a remarkable number of biological processes.There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of highthroughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be "hot spots" for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

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“…Thus, detectable current changes can be observed when the target analyte (e.g., RNA) is trapped inside the pore after hybridization with a target specific capture probe . Until now, only miRNAs have been interrogated via nanopores . Wang et al developed a unique nanopore sensor using α‐hemolysin protein pore ( Figure ).…”

Section: Rna Biomarker Detection Technologiesmentioning

confidence: 99%

“…Additionally, the sensor was successfully tested to differentiate the relative levels of miR‐155 of healthy and lung cancer patients. A comparative analysis of the advantages, disadvantages, and analytical functionalities of the nanopore sensors along with other major RNA detection platforms is summeaized in Table 2 .…”

Section: Rna Biomarker Detection Technologiesmentioning

confidence: 99%

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

et al. 2017

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Ribonucleic acids (RNAs) are considered as effective and minimally invasive biomarkers for disease diagnosis and prognosis due to their critical role in the regulation of different cellular processes. Over the past several years, the rapid progress in RNA biomarker research has resulted in the development of a large number of high‐performance RNA‐detection methods. Most of these methods are based on molecular‐biology techniques such as quantitative reverse transcription polymerase chain reaction (RT‐qPCR), microarrays, and RNA sequencing. In recent years, considerable attention has also been dedicated to developing RNA biosensors, exploiting micro‐ and nanofabrication technologies, and various readout strategies, including electrochemical and optical transducers. Here, the recent developments of RNA biosensors are concisely reviewed with a special emphasis on electrochemical‐detection approaches. The following points are also highlighted: i) all the types of clinically relevant RNAs (mRNAs, miRNAs, lncRNAs) and their diagnostic and prognostic potential in cancer are outlined, ii) major challenges associated with current techniques are identified, followed by a critical analysis of how these challenges have been addressed by different biosensing approaches, and iii) the current requirements that still need to be met for effective screening of RNA biomarkers in both research and clinical settings.

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Studies of RNA Sequence and Structure Using Nanopores

Cited by 27 publications

References 59 publications

Directional translocation resistance of Zika xrRNA

Directional translocation resistance of Zika xrRNA

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

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