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Related Concept Videos

Riboswitches01:56

Riboswitches

Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
The aptamer has high specificity for a particular metabolite which allows riboswitches to specifically regulate...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Experimental RNAi02:15

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
Transcriptional Regulation: Riboswitches01:23

Transcriptional Regulation: Riboswitches

Riboswitches are RNA elements that regulate gene expression by altering their secondary structures in response to specific effector molecules. These elements, located in the leader regions of certain mRNAs, act as transcriptional regulators by toggling between alternative conformations to control downstream gene expression. Riboswitch-mediated regulation is a precise mechanism for modulating biosynthetic pathways, as exemplified by the riboflavin biosynthesis pathway in Bacillus...
RNA Interference01:23

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...

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RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes
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A versatile cis-blocking and trans-activation strategy for ribozyme characterization.

Andrew B Kennedy1, Joe C Liang, Christina D Smolke

  • 1Department of Bioengineering, Stanford University, 473 Via Ortega, MC 4201, Stanford, CA 94305, USA.

Nucleic Acids Research
|November 17, 2012
PubMed
Summary
This summary is machine-generated.

Researchers developed a gel-free method to efficiently produce full-length synthetic RNA for gene-regulatory devices. This new technique simplifies ribozyme characterization and advances the engineering of RNA-based control systems.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biochemistry

Background:

  • Ribozyme-based synthetic RNA control devices regulate cellular functions via environmental signals.
  • Accurate measurement of ribozyme cleavage rates is crucial for optimizing these gene-regulatory devices.
  • Generating full-length RNA for characterization is challenging due to in vitro cleavage during transcription.

Purpose of the Study:

  • To develop an efficient, gel-free method for scalable generation of functional full-length ribozyme-encoding RNA.
  • To establish a rapid, label-free assay for real-time monitoring of ribozyme cleavage.
  • To advance the characterization and engineering of ribozyme-based gene-regulatory devices.

Main Methods:

  • A two-step gel-free process involving cis-blocking and trans-activation was developed for RNA generation.
  • The strategy was validated using natural and synthetic ribozymes.
  • Surface plasmon resonance (SPR) was employed to create a label-free, real-time cleavage assay.

Main Results:

  • The developed method enables scalable production of full-length ribozyme-encoding RNA without laborious gel separation.
  • Cleavage rate constants obtained using the new method are comparable to traditional approaches.
  • The SPR assay provides continuous, real-time monitoring of ribozyme activity.

Conclusions:

  • The novel cis-blocking and trans-activation strategy offers a simplified and scalable approach to generating functional full-length ribozyme RNA.
  • The SPR-based assay complements this strategy, enhancing the characterization of ribozyme devices.
  • These advancements will accelerate the design and optimization of synthetic RNA control systems.