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the known flanking regions in the case of DNA, or reverse transcribed and
then PCR amplified for RNA molecules, and the process is repeated until a
satisfactory level of enrichment is attained.
In order to isolate sequence-independent self-cleaving molecules using
an in vitro selection, a fundamentally different approach is required to amplify
active sequences. Because self-cleaved RNAs are covalently bisected, iso-
lated sequences can no longer be directly amplified for use in subsequent
rounds of selection unless the cleavage site is known a priori . This dilemma
can be overcome in one of two ways. First, the RNA library can be circu-
larized following transcription but prior to its incubation in media that sup-
port self-cleavage. After self-scission, the RNA will exist as an intact, but
linear, molecule that can be enzymatically ligated and then amplified for
use in subsequent rounds of the selection. 93,94
Alternatively, the DNA can be circularized, and transcription of such a
template enables a rolling-circle mechanism to yield concatemeric RNA
molecules that each contains multiple copies of a single pool RNA
sequence. 2,95-97 In all but monomer length transcripts, both flanking
primers and promoter regions will be present in the proper orientations
for productive reverse transcription and amplification to regenerate the
DNA pool for subsequent rounds of selection.
The human CPEB3 ribozyme was isolated using the rolling-circle tran-
scription technique on a circular pool constructed from digested human
genomic DNA ( Fig. 4.4 ). 2 These 150 nucleotide genomic DNA fragments
were ligated to primers, amplified using biotinylated forward primers and 5 0
phosphorylated reverse primers, immobilized on a streptavidin column, and
subsequently denatured to elute the phosphorylated reverse strand. The
single-stranded sequences were splint-ligated, and the splint oligonucleotide
was extended by DNA polymerase to yield nicked, double-stranded DNA
(dsDNA) circles for use in rolling-circle transcriptions.
To select for self-cleaving ribozymes, the dsDNA pool was transcribed
in a rolling-circle fashion to yield the multimeric RNA. These
concatemers were gel-purified, incubated for 1 h in a buffer conducive to
self-cleavage (10 mM Mg 2 รพ ), and the bands corresponding to self-cleaved
dimeric RNA species were gel-purified again. Reverse transcription
and PCR amplification with the biotinylated and phosphorylated
primers restored the DNA pool for subsequent rounds of the experiment.
After several cycles of selection, robust self-cleaving sequences came to
dominate the population, among them the HDV-like fold found in the
CPEB3 gene.
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