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Related Concept Videos

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...
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...
Allosteric Regulation01:08

Allosteric Regulation

Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
Allosteric Regulation01:08

Allosteric Regulation

Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...

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Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction
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Selecting allosteric ribozymes.

Nicolas Piganeau1

  • 1Institut für Biochemie und Molekularbiologie, Universität Hamburg, Hamburg, Germany. nicolas.piganeau@chemie.uni-hamburg.de

Methods in Molecular Biology (Clifton, N.J.)
|February 9, 2012
PubMed
Summary
This summary is machine-generated.

Scientists designed allosteric ribozymes that act as molecular switches, responding to specific drugs like doxycycline. This breakthrough enables new tools for gene regulation and biosensing applications.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biochemistry

Background:

  • Allosteric ribozymes are RNA molecules that can be engineered to bind specific targets.
  • These ribozymes can function as synthetic gene regulators or biosensors.
  • In vitro selection is a powerful technique for discovering functional RNA molecules from large libraries.

Purpose of the Study:

  • To develop a protocol for the de novo selection of allosteric self-cleaving ribozymes.
  • To create ribozymes that specifically respond to drugs of choice.
  • To demonstrate the selection of hammerhead ribozymes sensitive to doxycycline and pefloxacin.

Main Methods:

  • Utilized in vitro selection strategies to isolate allosteric ribozymes from extensive molecular libraries (up to 10^15 molecules).
  • Applied the developed protocol to select for hammerhead ribozymes with specific drug responses.
  • Characterized the selected ribozymes for their sensitivity to doxycycline and pefloxacin in the sub-micromolar range.

Main Results:

  • Successfully selected allosteric hammerhead ribozymes that are specifically inhibited by doxycycline and pefloxacin.
  • Achieved high sensitivity, with inhibition occurring in the sub-micromolar concentration range.
  • Demonstrated that these selected ribozymes can be converted into aptamers by introducing a point mutation.

Conclusions:

  • The described in vitro selection strategy is effective for de novo selection of drug-specific allosteric ribozymes.
  • Selected ribozymes offer potential applications as synthetic gene expression regulators and biosensors.
  • The ability to convert these ribozymes into aptamers expands their utility in molecular biology and diagnostics.