Biomolecules lectures on In vitro selection (SELEX) Apr. 2, 2001

Putting the RNA world to work

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

In vitro selection or SELEX

Gold and Tuerk (1990) Science 249,505-510
Ellington and Szostak (1990) Nature 346, 818-822.
Start with Synthesized oligo:

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)

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.

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 non-random sequence

"Sexual" PCR

Stemmer (1994) PNAS 91, 10747-51
Idea similar to mutagenic PCR, but mix and match variant homologous sequences with low activity by recombination

==>In vitro recombination