Combinatorial methods and activity selection
RNA World
idea that RNA might have been first molecule to self replicate
- can encode it's own replication (proteins can't)
- has ability to catalyze chemical reactions (DNA can't)
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:
- need way to select for molecules you want and get rid of those you don't:
- protein binding (aptamers)
- column binding " "
- self-cleavers or -ligaters (--> change in gel mobility)
- The strategy
- Transcribe RNA
- Select active molecules
- PCR amplify active molecules (reverse transcribe, then PCR)
- repeat many cycles
--> collection of molecules selected to do what you want.
Dilemma
- longer random sequence more likely to have wanted functional groups
- but as RNA gets longer, takes more sequence to represent all possible sequences.
random sequence # possible sequences
20-mer 4e20 =1.1X10e12 =1.8 pmoles = 12 ng
30-mer 4e30 =1.1X10e18 =1.8 umoles = 18 mg
40-mer 4e40 =1.2X10e24 =2 moles! = 26 kg!
165-mer 4e165 =2.2X10e99
6 orders of magnitude/10 additional nts.
RNA ligase selection
- Selected for RNAs that could ligate a tagged oligo to their own 5' end.
- Strategy for selection


- 10e15 seqs: wound up using 1.6 mg of RNA in first round.
- Ligation: Cycled temp. to sample different conformations
- Select for attachment of substrate oligo.
- Used different tag sequences in case of chance affinity of "enzyme"
for tag. or resin. (BIG potential problem in selection schemes)
- Elute.
- Selective PCR (only ligated sequences amplified)
- Reconstructive PCR -- get rid of substrate seqs. add back T7 promoter
- Transcribe --> reconstructed RNA enzyme ready to react in next round.
- Mutagenic PCR in middle rounds. Allows suboptimal RNAs to evolve into
more efficient ones
- Reduce reaction time, Mg --> more stringent selection
Phylogenetic analysis to determine structure of ligase:
- Resynthesized ligase sequence with "doped" oligonucleotides
- Select for activity and sequence individual active RNAs
- Align sequences and analyze or conserved regions and covariations
Biochemical Characterization of enzyme activity
Reconfigure RNA into enzyme and substrate RNAs
run products on gel to characterize:

- analyzed products by digestion and TLC
- revealed that only class I enzyme was forming 3'-5' linkages found in
cellular RNA and DNA polymers
- Others were 2'-5' links.
- This means the reverse transcriptase used to amplify RNA was able to
use 2'-5' linkage RNAs as template.
- This ligase has been used as a starting point for selection of RNA polymerase
activity that can faithfully synthesize copies of RNA templates.
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