Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

McKinley, Lauren N.; Kern, Reuben G.; Assmann, Sarah M.; Bevilacqua, Philip C.

doi:10.1021/acs.biochem.3c00506

Cited by 2 publications

(2 citation statements)

References 71 publications

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“…These findings have implications for other ribozyme cleavage measurements that require RNA purification/extraction and reverse transcription, such as RNA-seq (32,(38)(39)(40)(41)(42)(43). In particular, reports of context-dependent ribozyme cleavage that used different measurement techniques in vitro and in cells and found differences in activity may warrant additional examination (25,45,46). It is worth noting that HDV-like ribozymes are very stable and retain catalytic activity at high temperatures, so they may be uniquely susceptible to unwanted activity during sample denaturation and reverse transcription compared to other ribozyme folds (48,68).…”

Section: Discussionmentioning

confidence: 90%

“…These applications span in vitro transcription (IVT), cell-free lysates, and prokaryotic and eukaryotic cells, with each environment presenting unique conditions that could influence ribozyme activity (16)(17)(18)(19). Further, ribozymes are often used in many different genetic contexts, i.e., with different sequences flanking the ribozyme, and each new context has the potential to introduce alternative folds that change activity (20)(21)(22)(23)(24)(25). Together, these differences in ribozyme activity across genetic and environmental contexts represent context-dependent effects (Figure 1A) that need to be measured when developing new ribozyme-based technologies or discovering new ribozyme sequences.…”

Section: Introductionmentioning

confidence: 99%

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Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

Alperovich,

Vasilyeva,

Schaffter

2024

Preprint

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Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.

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Section: Discussionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

Alperovich,

Vasilyeva,

Schaffter

2024

Preprint

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Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections

McKinley,

Meyer,

Sebastian

et al. 2024

Preprint

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Twister ribozymes are an extensively studied class of nucleolytic RNAs. Thousands of natural twisters have been proposed using sequence homology and structural descriptors. Yet, most of these candidates have not been validated experimentally. To address this gap, we developed CHiTA (Cleavage High-Throughput Assay), a high-throughput pipeline utilizing massively parallel oligonucleotide synthesis and next-generation sequencing to test putative ribozymesen massein a scarless fashion. As proof of principle, we applied CHiTA to a small set of known active and mutant ribozymes. We then used CHiTA to test two large sets of naturally occurring twister ribozymes: over 1, 600 previously reported putative twisters and ∼1, 000 new candidate twisters. The new candidates were identified computationally in ∼1, 000 organisms, representing a massive increase in the number of ribozyme-harboring organisms. Approximately 94% of the twisters we tested were active and cleaved site-specifically. Analysis of their structural features revealed that many substitutions and helical imperfections can be tolerated. We repeated our computational search with structural descriptors updated from this analysis, whereupon we identified and confirmed the first intrinsically active twister ribozyme in mammals. CHiTA broadly expands the number of active twister ribozymes found in nature and provides a powerful method for functional analyses of other RNAs.GRAPHICAL ABSTRACT

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Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

Cited by 2 publications

References 71 publications

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections

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