High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase

Ditzler, Mark A.; Lange, Margaret J.; Bose, Debojit; Bottoms, Christopher A.; Virkler, Katherine F.; Sawyer, Andrew W.; Whatley, Angela S.; Spollen, William G.; Givan, Scott A.; Burke, Donald H.

doi:10.1093/nar/gks1190

Cited by 57 publications

(104 citation statements)

References 36 publications

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“…Due to this complexity, interpreting ligated P2 sequences enriched or depleted from the initial P2-5N library as being more or less fit for ligation requires validating data to avoid misleading conclusions (Ditzler et al 2013;Kupakuwana et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

et al. 2016

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The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

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Section: Discussionmentioning

confidence: 99%

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

et al. 2016

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“…3). While traditional selection approaches often rely upon overselection to facilitate finding an aptamer with limited sequence information, preservation of the diversity of winners and sequence analysis by NGS allows for a more thorough analysis of conservation and covariation patterns, aiding in determining consensus aptamer sequences 30,31 . Similar results were observed in the GR-scaffolded L-DOPA selection (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

et al. 2017

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Allosteric RNA devices are increasingly viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small molecule binding RNA aptamers that robustly function in the cellular environment. While aptamers can be raised against nearly any desired target by in vitro selection, many cannot be easily integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary and tertiary structural scaffolds derived from biologically active riboswitches and small ribozymes. Applied to neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yields easily identifiable and characterizable aptamers predisposed for coupling to readout domains to engineer nucleic acid sensory devices that function in vitro and in the cellular context.

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“…Another bioinformatics pipeline was used by Ditzler et al to find a better RNA inhibitor of HIV reverse transcriptase reanalyzing a SELEX experiment previously performed in 1996 [31,32]. Around 1,000,000 sequences from round 14 were analyzed.…”

Section: Identification Of Secondary Structure Motifsmentioning

confidence: 99%

“…Ditzler et al used such strategy to select aptamers with a slower dissociation rate [32]. Following the study previously described to identify RNA aptamers against the reverse transcriptase (RT) of HIV, they compared the frequency of sequences in a pool that underwent one round of SELEX with increasing stringency.…”

Section: Study Of Specificity and Fitness Landscapesmentioning

confidence: 99%

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

2016

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Aptamers are identified through an iterative process of evolutionary selection starting from a random pool containing billions of sequences. Simultaneously to the amplification of high-affinity candidates, the diversity in the pool is exponentially reduced after several rounds of in vitro selection. Until now, cloning and Sanger sequencing of about 100 sequences was usually used to identify the enriched candidates. However, High-Throughput Sequencing (HTS) is now extensively used to replace such low throughput sequencing approaches. Providing a deeper analysis of the library, HTS is expected to accelerate the identification of aptamers as well as to identify aptamers with higher affinity. It is also expected that it can provide important information on the binding site of the aptamers. Nevertheless, HTS requires handling a large amount of data that is only possible through the development of new in silico methods. Here, this review presents these different strategies that have been recently developed to improve the identification and characterization of aptamers using HTS.

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High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase

Cited by 57 publications

References 36 publications

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

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