TU Darmstadt / ULB / TUbiblio

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers.

König, Julian and Julius, Christian and Baumann, Sebastian and Homann, Matthias and Göringer, H. Ulrich and Feldbrügge, Michael (2007):
Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers.
13, In: RNA (New York, N.Y.), (4), pp. 614-22, ISSN 1355-8382, [Article]

Abstract

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.

Item Type: Article
Erschienen: 2007
Creators: König, Julian and Julius, Christian and Baumann, Sebastian and Homann, Matthias and Göringer, H. Ulrich and Feldbrügge, Michael
Title: Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers.
Language: English
Abstract:

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.

Journal or Publication Title: RNA (New York, N.Y.)
Volume: 13
Number: 4
Divisions: 10 Department of Biology > Postranscriptional Gene Regulation and RNA Therapeutics
?? fb10_mikrobiologie ??
10 Department of Biology
Date Deposited: 03 Nov 2011 13:01
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)

View Item View Item