Design and analysis of hammerhead ribozyme activity against an artificial gene target

James R. Carter; Pruksa Nawtaisong; Velmurugan Balaraman; Malcolm J. Fraser

doi:10.1007/978-1-62703-730-3_5

Design and analysis of hammerhead ribozyme activity against an artificial gene target

James R. Carter
, Pruksa Nawtaisong
, Velmurugan Balaraman
, Malcolm J. Fraser

Biochemistry, Chemistry & Physics

University of Notre Dame

Research output: Contribution to book or proceeding › Chapter › peer-review

Abstract

In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

Original language	English
Title of host publication	Therapeutic Applications of Ribozymes and Riboswitches
Subtitle of host publication	Methods and Protocols
Publisher	Humana Press Inc.
Pages	57-66
Number of pages	10
ISBN (Print)	9781627037297
DOIs	https://doi.org/10.1007/978-1-62703-730-3_5
State	Published - 2014

Publication series

Name	Methods in Molecular Biology
Volume	1103
ISSN (Print)	1064-3745

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Scopus Subject Areas

Molecular Biology
Genetics

Keywords

Catalysis
Dengue
Electrophoresis
Flavivirus
In vitro transcription
RNA
Ribozyme

Access to Document

10.1007/978-1-62703-730-3_5

Cite this

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title = "Design and analysis of hammerhead ribozyme activity against an artificial gene target",

abstract = "In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.",

keywords = "Catalysis, Dengue, Electrophoresis, Flavivirus, In vitro transcription, RNA, Ribozyme",

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series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

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}

Design and analysis of hammerhead ribozyme activity against an artificial gene target. / Carter, James R.; Nawtaisong, Pruksa; Balaraman, Velmurugan et al.
Therapeutic Applications of Ribozymes and Riboswitches: Methods and Protocols. Humana Press Inc., 2014. p. 57-66 (Methods in Molecular Biology; Vol. 1103).

Research output: Contribution to book or proceeding › Chapter › peer-review

TY - CHAP

T1 - Design and analysis of hammerhead ribozyme activity against an artificial gene target

AU - Carter, James R.

AU - Nawtaisong, Pruksa

AU - Balaraman, Velmurugan

AU - Fraser, Malcolm J.

PY - 2014

Y1 - 2014

N2 - In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

AB - In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

KW - Catalysis

KW - Dengue

KW - Electrophoresis

KW - Flavivirus

KW - In vitro transcription

KW - RNA

KW - Ribozyme

UR - https://www.scopus.com/pages/publications/84892873362

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DO - 10.1007/978-1-62703-730-3_5

M3 - Chapter

C2 - 24318886

AN - SCOPUS:84892873362

SN - 9781627037297

T3 - Methods in Molecular Biology

SP - 57

EP - 66

BT - Therapeutic Applications of Ribozymes and Riboswitches

PB - Humana Press Inc.

ER -

Design and analysis of hammerhead ribozyme activity against an artificial gene target

Abstract

Publication series

UN SDGs

Scopus Subject Areas

Keywords

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