23 510 Finding RNA Sequences That Interact with Other Molecules

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Chapter 5 / Molecular Tools for Studying Genes and Gene Activity
Thus, the protein–protein interactions suggested by such
an assay should be verified with a direct assay, such as
immunoprecipitation.
P T7
SUMMARY Protein–protein interactions can be de-
tected in a number of ways, including immunoprecipitation and yeast two-hybrid assay. In the latter
technique, three plasmids are introduced into yeast
cells. One encodes a hybrid protein composed of
protein X and a DNA-binding domain. The second
encodes a hybrid protein composed of protein Y
and a transcription-activating domain. The third
has a promoter-enhancer region linked to a reporter
gene such as lacZ. The enhancer interacts with the
DNA-binding domain linked to protein X. If proteins X and Y interact, they bring together the two
parts of a transcription activator that can activate
the reporter gene, giving a product that can catalyze a
colorimetric reaction. If X-gal is used, for example,
the yeast cells will turn blue.
5.10 Finding RNA Sequences
That Interact with Other
Molecules
DNAs
(a)
Transcribe
(d) PCR
cDNAs
RNAs
(b)
Select by binding
to a target molecule
(c) Reversetranscribe
Selected
RNAs
Figure 5.41 SELEX. Start with a large collection of DNAs (top) that
have a random sequence (blue) flanked by constant sequences (red).
(a) Transcribe the DNA pool to produce a pool of RNAs that also
contain a random sequence flanked by constant sequences.
(b) Select for aptamers by affinity chromatography with the target
molecule. (c) Reverse-transcribe the selected RNAs to produce a
pool of cDNAs. (d) Amplify the cDNAs by PCR, using primers
complementary to the constant regions at the ends of the DNAs. This
cycle is repeated several times to enrich the aptamers in the pool.
SELEX
Functional SELEX
SELEX (systematic evolution of ligands by exponential
enrichment) is a method that was originally developed to
discover short RNA sequences (aptamers) that bind to
particular molecules. Figure 5.41 illustrates the classical
SELEX procedure. One starts with a pool of PCR-amplified
synthetic DNAs that have constant end regions (red), but
random central regions (blue) that can potentially encode
over 1015 different RNA sequences. In the first step, these
DNAs are transcribed in vitro, using the phage T7 RNA
polymerase, which recognizes the T7 promoter in the upstream constant region of every DNA in the pool. In the next
step, the aptamers are selected by affinity chromatography
(this chapter), using a resin with the target molecule immobilized. The selected RNAs bind to the resin and then can be
released with a solution containing the target molecule.
These selected RNAs are then reverse-transcribed to yield
double-stranded DNA, which is then subjected to PCR, using primers specific for the DNAs’ constant ends.
One round of SELEX yields a population of molecules
only partially enriched in aptamers, so the process is repeated several more times to produce a highly enriched population of aptamers. SELEX has been extensively exploited
to find the RNA sequences that are contacted by proteins. It
is extremely powerful in that it finds a few aptamers among
an astronomically high number of starting RNA sequences.
Functional SELEX is similar to classical SELEX in that it
finds a few “needles” (RNA sequences) in a “haystack” of
starting sequences. But instead of finding aptamers that bind
to other molecules, it finds RNA sequences that carry out, or
make possible, some function. With simple binding, selection
is easy; it just requires affinity chromatography. But selection
based on function is trickier and requires creativity in designing the selection step. For instance, the first functional
SELEX procedures detected a ribozyme (an RNA with enzymatic activity), and this ribozyme activity altered the RNA
itself to allow it to be amplified. One simple example is a
ribozyme that can add an olignucleotide to its own end.
This activity allowed the investigators to supply an oligonucleotide of defined sequence to the ribozyme, which then
added this tag to itself. Once tagged, the ribozyme becomes
subject to amplification using a PCR primer complementary
to the tag.
A pool of random RNA sequences may not contain any
RNAs with high activity. But that problem can be overcome by carrying out the amplification step under mutagenizing conditions, such that many variants of the mildly
active sequences are created. Some of these will probably
have greater activity than the original. After several rounds
of selection and mutagenesis, RNAs with very strong enzymatic activity can be produced.