Combinatorial methods and activity selection

RNA World

idea that RNA might have been first molecule to self replicate

In vitro selection or SELEX

Putting the RNA world to work

Take advantage of having active groups and genetic info on same molecule.

Gold and Tuerk (1990) Science 249,505-510 (SELEX)
Ellington and Szostak (1990) Nature 346, 818-822. (in vitro selection)

Start with Synthesized oligonucleotide containing random sequence:

Dilemma

RNA ligase selection

 

Phylogenetic analysis to determine structure of ligase:

Biochemical Characterization of enzyme activity

Reconfigure RNA into enzyme and substrate RNAs
run products on gel to characterize:

Secondary structure of other RNA ligases (2'-5')

Ways around sequence complexity problem

Probably don't need all seq. at all positions

Mutagenic PCR

use Mn and altered dNTP concs. to increase mutation rate of PCR
So molecule not present initially may arise from earlier sequence with lower activity
"In vitro evolution"

Can start with nonrandom sequence

Begin with RNA that performs similar function to that desired.
Randomize only part of the molecule likely to be critical for function

--> optimized version of RNA based on existing prototype
also shows variability of less important parts

 

DNA shuffling

Stemmer (1994) PNAS 91, 10747-51
Idea similar to mutagenic PCR, but mix and match variant homologous sequences with low activity by recombination
digest DNAs that are related but different to random short oligos with DNAseI
mix and anneal
select for full-length molecules by PCR of constant flanking regions
==>In vitro recombination


An example

RNAseP protein similar in structure to EF-G.

Use PCR to make chimeric proteins:

Tomorrow: Data mining lab ED305P

Jim Nolan
Rm. 6014
jnolan@tulane.edu