Searching for ribozymes with new and improved properties

The technique of in vitro selection was used to learn more about the properties of RNA sequence space. In Chapter 1 I investigated how readily ribozymes with new catalytic activities and folds could arise from an existing ribozyme scaffold. In vitro selection was used to isolate 23 kinase ribozymes from a pool of more than 104 variants of an aminoacylase parent ribozyme. The density of kinase ribozymes in the pool increased dramatically as the mutational distance from the starting ribozyme increased, suggesting a need to escape the fold of the parent. Consistent with this idea, the folds of two kinases characterized in detail were different from that of the starting aminoacylase scaffold. In Chapter 2 I1 investigated the extent to which one of these kinase ribozymes could be optimized using a method of recombination called synthetic shuffling. Point mutations from previously isolated sequence variants of this ribozyme were shuffled in more than 1014 different combinations, and active variants isolated by in vitro selection. The rate of the most efficient ribozyme identified was 30-fold faster than that of the most efficient ribozyme used to build the pool, with a second order rate enhancement approaching 100°- fold.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Curtis, Edward A. (Edward Arthur)

Advisor dc:contributor.advisor

• David P. Bartel.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/37455
• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Identifier URI

http://dspace.mit.edu/handle/1721.1/37455

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/37455