Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium

Naghdi, Mohammad Reza; Boutet, Emilie; Mucha, Clarisse; Ouellet, Jonathan; Perreault, Jonathan

doi:10.3390/ncrna6010014

Cited by 3 publications

(4 citation statements)

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“…The previously published A6C mutation in the core of a HHR ( Fig. 5 A) was shown to have a k obs of 0.18 min −1 in the presence of Mn 2+ ( Naghdi et al 2020 ), much faster than the 0.005 min −1 of the example described above ( Fig. 1 A).…”

Section: Resultsmentioning

confidence: 47%

“…( D ) As in Figure 5 C, interpolated fluorescence values were mapped using the standard curve to concentrations of released output strand. It should be noted that the ribozyme adapted from Naghdi et al (2020) was described as having a pseudoknot (as was the original version of this HHR described previously [ Perreault et al 2011 ] which had an A at position 6 from the core, instead of a C in this “Mn 2+ -HHR”). However, secondary structure prediction also suggests an alternative structure ( Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A second ribozyme example, which cleaves in the presence of Mn 2+ (but not Mg 2+ ), was selected from the literature ( Naghdi et al 2020 ). The fact that this ribozyme is inactive in the presence of Mg 2+ allowed us to prepare self-cleaving ribozymes easily for assays in TMSDR.…”

Section: Methodsmentioning

confidence: 99%

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Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes

Kapadia

Kharma

Davis

et al. 2021

RNA

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This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5′-end, while the other strand is labelled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

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Section: Resultsmentioning

confidence: 47%

Section: Resultsmentioning

confidence: 99%

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Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes

Kapadia

Kharma

Davis

et al. 2021

RNA

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“…Earlier, the 2′-hydroxyl group of G8 was thought to be the acid in this acid-base catalysis. However, a recent study reports that Mn 2+ -bound water is the general acid during cleavage [25].…”

Section: Mechanism Of Catalysis In Ribozymes: Ribozymes Perform Rever...mentioning

confidence: 99%

Ribozymes as Therapeutic Agents against Infectious Diseases

Wong¹,

Shahid²,

Tan³

2023

RNA Therapeutics - History, Design, Manufacturing, and Applications

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Ribozymes, also known as RNA enzymes, are catalytic RNA molecules capable of cleaving specific RNA sequences, leading to decreased expression of targeted genes. Recent studies suggest their role in cancer therapeutics, genetic diseases and retroviral infections. This book chapter will focus on ribozymes acting as therapeutic agents against infectious diseases caused by viral and bacterial pathogens. Firstly, we will introduce a brief history of ribozymes and a general overview of ribozymes and their characteristics. Next, different types of ribozymes will be explored regarding their targets and mechanisms of action. After that, ribozymes specific to viral and bacterial infections will be explored. We will briefly discuss the current status of ribozymes as therapeutic agents. Finally, the roadblock and challenges ribozymes face before being developed into therapeutic agents—such as their delivery and efficacy issues—will be discussed.

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Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection

Brill,

Nurmi,

2024

ChemBioChem

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The Hammerhead Ribozyme (HHR) is a ubiquitous RNA enzyme that catalyzes site‐specific intramolecular cleavage. While mutations to its catalytic core have traditionally been viewed as detrimental to its activity, several discoveries of naturally occurring variants of the full‐length ribozyme challenge this notion, suggesting a deeper understanding of HHR evolution and functionality. By systematically introducing mutations at key nucleotide positions within the catalytic core, we generated single‐, double‐, and triple‐mutation libraries to explore the sequence requirements and evolution of a full‐length HHR. In vitro selection revealed many novel hammerhead variants, some of which possess mutations at nucleotides previously considered to be essential. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids.

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Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium

Cited by 3 publications

References 42 publications

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes

Ribozymes as Therapeutic Agents against Infectious Diseases

Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection

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