Download RNA Ligands to Bacteriophage T4 DNA Polymerase

Systematic Evolution of Ligands
by Exponential
Enrichment: RNA Ligands to
Bacteriophage
T4 DNA Polymerase
CRAIG TUERK AND LARRY GOLD
So what was being studied?
• The interaction of bacteriophage T4 DNA
polymerase (gp43) and the ribosome
binding site of the mRNA that it encodes
But a bit of background first…
• Binding gp43 overlapping the SD
sequences prevents translation and
promotes autogenous regulation
• The minimum target size of the RNA is
36nt which includes a 5bp helix and 8nt
loop
This is also
conserved in
phage RB69
To investigate what is
responsible for this loop bias,
the researches created the
SELEX method to select
preferred binding sequences
from a random sequence pool
But what the heck is SELEX?
• Sytematic Evolution of Ligands by
EXponential enrichment.
• The method relies on mechanisms that are
often ascribed to evolution to accomplish
its goal.
– Variation
– Selction
– Replication
Variation
Selection
Replication
Creation of Variant Sequences
• Created a 110nt ssDNA transcript by ligating three
oligonucleotides together and two bridging oligos
• Oligo 4 was randomized for the 8nt of the loop
sequence (AAUAACUC), creating 65,536
sequences
• WT sequence was also engineered and used for
comparison with the results.
Sequence showing
variation in the
conserved 8nt loop
domain vs. WT
sequence
Comparison of dissociation
constants
• Kd for WT:gp43 = 5x10^-9
• Kd for variant sequences = 3.2x10^-7
– WT binding is about 60x stronger than variant
sequences, but only 100x stronger than
nonspecific binding
So this leads us to a goal
• To enrich for the highest affinity RNA
ligands to gp43
Experiment
• Three experiments were carried out at different
concentrations ratios of RNA to gp43.
• A=10
• B=1000
• C=100
• RNA was added to gp43 in excess of binding
sites so that competitive binding occurred.
• Amount of RNA retrieved was roughly equal to
the amount of gp43 in the reaction
Selection was carried
out by allowing gp43
to bind RNA ligand.
Complex was
purified by capture in
a nitrocellulose filter.
-Purified RNA was
subjected to reverse
transcriptase and
PCR amplification
-Amplified cDNA was
converted to RNA
with T7 transcription
What happened?
• After four rounds of selection the labeled
products exhibited binding that was
indistinguishable from WT binding
– Though it is not explained HOW they know this
• Three rounds of B were gel purified and
sequenced
• The final round of all the experiments were
purified and sequenced.
Shows the evolution of the RNA ligand
Shows a “consensus sequence”
develop in the batches
• WT sequence is present in the consensus
sequence
• Using quantification data, it is shown there
is a slight bias for the WT (data not shown)
So what sequences are
represented?
• Individual sequences from B were cloned
into pUC18 (plasmid). Bam H1 and Hind III
sequences were added for entry.
• 20 individual clones were selected and
sequenced
2 predominant sequences found
• WT AAUAACUC was found nine times
• AGCAACCU was found eight times
– Will now be referred to as Major Variant, MV
• 3 other single transition mutants were found
Additional binding experiment
• Sequences were
excised from plasmid
and filtered as before
on nitrocellulose.
There is a correlation
between selected
abundance and
binding affinity.
Another additional binding
experiment
• Additional
experimenting (not
shown) determined that
WT and MV compete for
gp43 and that the
dissociation constant for
WT is twice that of MV.
Paradox?
• The loop of eight nucleotides is conserved,
even to the distant phage RB69, yet the
change in dissociation constant it brings
only represents a minimal change in
binding energy (2.5 kcal).
• Other mutations around this region can
provide a higher binding energy, but are
not as well conserved.
Models for RNA sequence
equivalence.
• Bound WT may contain an AC pair in its loop
• Extra base pair in MV may be denatured by
binding
• Each form may make equivalent but not identical
contacts with protein
• Each loop sequence may participate in unusual
base pair combinations with the operator folding
the RNA.
This would require further structural study
Evolution
• As stated before, SELEX uses
mechanisms associated with evolution,
and can be thought of as in vitro evolution.
– Binding is a selective pressure
– A vast number of variant RNA are sampled at
once and selected out
– Replication is carried out by PCR and T7 RNA
polymerase
Evolution of Ligand
• The authors predict that since the operatorrepressor relationship exists, that it must have
conferred at some time a selective advantage
• Other regions must have been under more
functional constraint. Location of the operator
did not infringe on coding regions or other
operator sequences
• T4 sequences tend to be AT rich, so operators
should be biased to be AU rich.
• Despite the constraints of experimental design
SELEX came up with the same answer as
natural evolution did.
So SELEX is pretty cool, isn’t it?
• The authors sure seem to think so
SELEX is a technique that combines genetic
selection and precision from biochemical
technique. It is somewhat simple to use in
that there needs to be no other scorable
phenotype than binding to partitioning
agent.
Application of SELEX
• Can be used to determine optimal binding
sequences for any nucleic acid binding protein
• Study interactions between TFs, repressors and
binding sequences
• Develop RNA molecules to interact with other
substrates giving examples of how the “RNA
world” may have functioned
– Could be a step toward replicating some of those
early enzymes
• Nucleic acid ligands as specific inhibitors of
proteins
• Evolution in a test tube