Rev and RRE interaction (Rev figure)
Take advantage of having active groups and genetic info on same molecule.
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.
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.
A
three-dimensional model of the Rev-binding element of HIV-1 derived from analyses
of aptamers.
Leclerc F, Cedergren R, Ellington AD
PDB
structure
Saw that the G:G pair in bold could be substituted by an A:A pair. modeled all possible G:G's wit same geometry as A:A.
Built model using MC-SYM modeling program.
Subsequent NMR structure (Battiste, et al.)validated in vitro selection and modeling result. Showed interaction of peptide with RNA
Noncanonical base pairs open up major groove from normal A-form geometry
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
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