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Selection of hammerhead ribozyme variants with low Mg2+ requirement: Importance of stem-loop II

Persson, Tina LU ; Hartmann, RK and Eckstein, F (2002) In ChemBioChem 3(11). p.1066-1071
Abstract
Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg2- concentrations were in vitro selected from a library with 78 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had e sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mm Mg2- concentration in concentration, 25 degreesC, pH 75). Interestingly, this very active variant was not identified by the selection procedure, Changing loop II from UUUU to GCAA or extension of stem 11 to three or four base pairs reduced the... (More)
Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg2- concentrations were in vitro selected from a library with 78 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had e sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mm Mg2- concentration in concentration, 25 degreesC, pH 75). Interestingly, this very active variant was not identified by the selection procedure, Changing loop II from UUUU to GCAA or extension of stem 11 to three or four base pairs reduced the cleavage rate by 2.0 - 2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so (or at low Mg2- concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degreesC and pH 7.5 in monophasic reactions, G ntext of several substrates with differences in the lengths of with endpoints between 75 and 90%. Such constructs promise to be advantageous for the inhibitation of gene expression in vivo. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
hammerhead ribozyme, metalloenzymes, ribozymes, RNA structure
in
ChemBioChem
volume
3
issue
11
pages
1066 - 1071
publisher
John Wiley & Sons
external identifiers
  • wos:000179111600004
  • pmid:12404631
  • scopus:0037021345
ISSN
1439-4227
DOI
10.1002/1439-7633(20021104)3:11<1066::AID-CBIC1066>3.0.CO;2-G
language
English
LU publication?
yes
id
e01c03d6-de8b-4ec7-9408-4732a944e9e8 (old id 324295)
date added to LUP
2007-11-08 14:38:54
date last changed
2017-05-21 03:38:44
@article{e01c03d6-de8b-4ec7-9408-4732a944e9e8,
  abstract     = {Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg2- concentrations were in vitro selected from a library with 78 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had e sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mm Mg2- concentration in concentration, 25 degreesC, pH 75). Interestingly, this very active variant was not identified by the selection procedure, Changing loop II from UUUU to GCAA or extension of stem 11 to three or four base pairs reduced the cleavage rate by 2.0 - 2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so (or at low Mg2- concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degreesC and pH 7.5 in monophasic reactions, G ntext of several substrates with differences in the lengths of with endpoints between 75 and 90%. Such constructs promise to be advantageous for the inhibitation of gene expression in vivo.},
  author       = {Persson, Tina and Hartmann, RK and Eckstein, F},
  issn         = {1439-4227},
  keyword      = {hammerhead ribozyme,metalloenzymes,ribozymes,RNA structure},
  language     = {eng},
  number       = {11},
  pages        = {1066--1071},
  publisher    = {John Wiley & Sons},
  series       = {ChemBioChem},
  title        = {Selection of hammerhead ribozyme variants with low Mg2+ requirement: Importance of stem-loop II},
  url          = {http://dx.doi.org/10.1002/1439-7633(20021104)3:11<1066::AID-CBIC1066>3.0.CO;2-G},
  volume       = {3},
  year         = {2002},
}