David P. Bartel
We are interested in the ability of RNA to catalyze reactions. We would like to know the roles of catalytic RNA in modern-day biology. We would also like to understand better the intrinsic catalytic abilities of RNA. For example, we want to know the types of reactions that RNA can catalyze and how easy it is for new RNA enzymes (ribozymes) to emerge. These issues are interesting in their own right and are also crucial for evaluating theories of life's origins and early evolution. Our studies often involve in vitro selection experiments. In vitro selection allows us to isolate very rare catalytic molecules from large libraries containing over 1015 different sequences.
Ribozymes that Catalyze Ligation and Polymerization. A set of ribozymes that promote an RNA ligation reaction was previously isolated from a large pool of random RNA sequences. We are examining the catalytic and structural features of these new ribozymes. This allows us to address questions relevant to the origin of biological catalysis, such as the abundance and distribution of catalysts in the set of all possible RNA sequences. Understanding of natural ribozymes is also enhanced when put into the broader context of what is possible when liberated from constraints imposed by history.
One of the ligases we are studying has a kcat exceeding one per second--a value greater than that of any other known RNA catalysts and approaching that of comparable protein enzymes. We are interested in understanding its unusually high rate of catalysis through more detailed kinetic and mechanistic analysis. For a ribozyme that emerged from random sequences, the secondary structure of this ligase is remarkably complex. We are probing the tertiary architecture of this ribozyme using chemical mapping, crosslinking, and ultimately, x-ray crystallography.
A version of our ligase ribozyme can catalyze RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of 3 to 6 mononucleotides. The ribozyme shows marked template fidelity; bases complementary to the template add up to 1000-fold more efficiently than do mismatched bases. The demonstration that RNA can synthesize RNA using the same reaction as that employed by protein enzymes supports theories of RNA self-replication during the early evolution of life. This ribozyme will serve as a new starting point for the evolution of RNAs with more efficient and extensive polymerization activity.
The Function of Spliceosomal RNAs. Nuclear pre-mRNA splicing is required for expression of most vertebrate genes. During splicing, introns are excised and exons are joined through successive phosphodiester transfer reactions. Splicing is performed by the spliceosome, a ribosome-sized complex which contains mostly protein but also includes, at various stages of the reaction, five small nuclear RNAs (snRNAs). It has been proposed that some of the snRNAs catalyze the transesterification reactions involved in splicing. However, when assayed without protein, the snRNAs have not shown any hint of catalytic activity. In collaboration with Phillip Sharp, we are generating catalytic RNAs that use the RNA components of the spliceosome thought to be involved in the catalytic steps of splicing.
We have constructed a library of more than 1014 different RNA molecules, in which the key sequences from snRNAs and pre-mRNA have been mutagenized at a low rate, linked to each other, and also linked to fully randomized segments. We are using in vitro selection to isolate members of the library that can carry out the second transesterification of splicing, resulting in exon ligation and intron release. Perhaps we will find entirely new ribozymes that have little relevance to mRNA splicing; if so, it will still be interesting to compare our new ribozymes with self-splicing introns. Alternatively, the critical RNA components of the spliceosome could largely retain their roles while the random-sequence portions of the molecule functionally substitute for spliceosome proteins. (These two possibilities will be distinguished by examining whether snRNA mutations known to effect mRNA splicing have analogous effects on the selected ribozymes.) If the spliceosome snRNA segments have largely retained their roles then we will be able to define the function of snRNAs during the catalytic steps of splicing.
Aptamers Insusceptible to Nuclease Degradation. In vitro selection can yield RNA and single-stranded DNA sequences that tightly bind a variety of large and small molecules. Some have proposed that these nucleic acid molecules with ligand-specific binding activity (called aptamers) could have diagnostic or therapeutic uses. A major drawback for useful application of aptamers is that normal RNA and DNA are susceptible to nucleases. L-DNA, the mirror-image form of natural DNA, is not recognized by nucleases. In collaboration with the Kim lab, we are developing a procedure to generate an L-DNA aptamer that binds and inhibits the peptide hormone, vasopressin.