Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method

Nucleic Acids Research
Michał ŁegiewiczJerzy Ciesiołka

Abstract

We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial 'structural plasticity' and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg2+, Ca2+ and Mn2+ ions. Thus, the functional properties of the variants ...Continue Reading

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Citations

Oct 16, 2007·Nucleic Acids Research·Atef NehdiJean-Pierre Perreault
Sep 26, 2013·PloS One·Danna G EickbushThomas H Eickbush
Jul 8, 2011·RNA Biology·Chiu-Ho T Webb, Andrej Lupták
Feb 11, 2014·Biochimica Et Biophysica Acta·Jerzy CiesiołkaWojciech Szczepanik
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Apr 4, 2015·Dalton Transactions : an International Journal of Inorganic Chemistry·Aleksandra KasprowiczJerzy Ciesiołka
May 5, 2016·Dalton Transactions : an International Journal of Inorganic Chemistry·K Stokowa-SołtysM Jeżowska-Bojczuk
Oct 25, 2008·Biochemistry·Mariola DutkiewiczJerzy Ciesiołka
Jul 9, 2021·Frontiers in Microbiology·Hans J NetterLilly K W Yuen
Sep 23, 2006·Science·Kourosh Salehi-AshtianiJack W Szostak

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Methods Mentioned

BETA
FRET
PCR
in vitro transcription
electrophoresis
cleavage assay

Software Mentioned

ImageQuant

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