Download In Vitro Selection of Metabolite-Dependent Self-Cleaving

In Vitro Selection of Metabolite-Dependent Self-Cleaving Ribozymes from the Mouse Genome
Tiffany Pham
Mentor: Andrej Luptak
Similar to enzymes, many RNAs have catalytic properties, such as peptide bond formation and self-cleaving.
Many self-cleaving ribozymes have already been found in viral, bacterial, and mammalian genomes. However,
only the glmS ribozyme, found in Gram-positive bacteria, has been found to self-cleave specifically in the
presence of a small-molecule co-factor. Since the glmS ribozyme may be involved in the gene expression of
the glmS gene, the discovery of additional metabolite-dependent self-cleaving RNAs might provide insight
into their seemingly important, but relatively unknown cellular roles. To perform an in vitro selection of such
ribozymes from the mouse genome, a DNA library has been constructed (a process monitored by gel
electrophoresis and radioactive labeling). This involved the ligation of double-stranded hairpin primers to
short genomic DNA fragments of unknown sequence and the subsequent removal of the single-stranded
loops, yielding genomic DNA flanked by known regions that enable amplification and circularization. Oneway extensions of this double-stranded library produced single-stranded DNA that was circularized by splint
ligation. This circular DNA will be made double-stranded and then transcribed to yield concatameric RNA
transcripts containing multiple copies of the same sequence. Upon incubation in the presence of mouse
small-molecule metabolites, the RNAs capable of self-cleavage will produce strands of progressively shorter
length. The transcripts containing the sequences that cleave will be purified using gel electrophoresis, reversetranscribed and amplified to afford the DNA pool for the next round of selection.