Our Science at a Glance
We are intensely interested in the biochemistry and biology of RNA. We study how cells and viruses use their own transcripts, we build RNA aptamers and ribozymes with new biochemical functions using in vitro selection (SELEX), and we take advantage of those new biochemistries to engineer new biologies. In short, we push back the boundaries of what is possible to achieve with RNA, and we seek to understand the underlying chemical and biological principles that imbue these molecules with their diverse functions.
Current projects address three Big Questions: How do you build a living bacterium from non-living parts? Can cells protect themselves from viruses by rebalancing which RNA-protein complexes are allowed to form? How can we program RNAs as "magic bullets" to study and manipulate cells that are otherwise difficult to target?
The Origins Project
During the 21st Century, Synthetic Life will be generated in a lab, and strong evidence of life will be found on planets in orbit around other stars. This is a very exciting time for origins research, and RNA will be a major player in those studies.
RNA is widely believed to have served a major role in the earliest evolution of Life on Earth, serving as a major storage form for heritable genetic information (before DNA) and as a major biocatalyst (before or along with protein enzymes). Metabolic ribozymes may still lie hidden within the genomes of modern organisms, and emergent life on other planets may experience chemical and evolutionary constraints similar to those that guided the emergence of Life on Earth. Furthermore, there is strong interest in constructing fully-artificial cells to model incipient life. However, there is little know about RNA's ability to catalyze chemical transformations involving small molecule metabolites.
A current question is to delineate the relationships among RNA enzyme mechanisms, SELEX library designs and fitness landscapes for phosphoryl transfer ribozymes. Metabolite phosphorylation is critical to biosynthetic and catabolic reactions and other cellular processes. Early life forms able to channel free energy from the environment into phosphorylated metabolites (blue in Fig. 1) would have been at a competitive advantage. Metabolite phosphorylation is therefore likely to have been among the first enzymatically catalyzed reactions. We have studied several ribozymes that phosphorylate RNA and DNA polymers. There is strong evidence that phosphorylation of small molecule metabolites should also be within reach of ribozyme catalysis, and a major effort is to generate and characterize such RNAs.
The Viral Proteins & RNA Project
Viruses are both a major threat to human health and elegant tools for restoring health. We study how RNA-protein interactions determine viral processes, such as the selective encapsidation of genome and certain cellular RNAs, and also how viral replication can be suppressed with aptamers that compete with normal RNA-protein interactions. We also engineer lentiviral vectors for delivering gene therapy agents.
The reverse transcriptase (RT) of human immunodeficiency virus (HIV-1) copies the viral RNA genome into DNA for insertion into the host chromosome. Aptamers that bind RT compete with viral genome for access to RT, thus preventing viral replication. In cell culture, viral loads drop several orders of magnitude to below detection levels in aptamer-expressing cells, but are unaffected in cells that express control RNAs.
A major push is to evaluate and optimize these aptamers for pre-clinical development for gene therapies, which we are pursuing along three lines: First, we are evaluating the anti-HIV efficacy of 1st-generation aptamers and expression vectors in hematopoietic stem cells (HSC) and in humanized mice. These are among the best animal models for pre-clinical testing of anti-HIV agents. Second, we are developing 2nd-generation aptamers and expression vectors. We are testing the hypothesis that broad-spectrum aptamers that recognize RT from phylogenetically diverse virus will be less susceptible to escape mutations than will aptamers that recognize RT from only one or a few viral clades. To improve expression vectors, we are optimizing engagement of the cell machinery involved in RNA expression, transport and accumulation. Third, we are seeking to understand and overcome HIV's capacity to evolve resistance to aptamers. We have developed innovative techniques that allow us to selecting for the emergence of aptamer-resistant HIV strains. At the same time, we are engineering aptamers with a "high genetic barrier to resistance," as these are the most likely to sustain a robust antiviral state in patients receiving aptamer therapies.
The Special Delivery Project
Aptamer binding specificity can be programmed to develop "smart drugs" for cell-targeted RNA therapies. We are currently pursuing four questions in this area. First, we are combining alternate positive/negative selection cycles with high throughput sequencing and informatics filters to identify aptamers that differentiate lymphatic cells and stem cells in various stages of differentiation or infection. Second, we are engineering those aptamers to deliver reagents that modulate expression of specific subsets of genes within the target cells, thus making it possible to study the roles of those genes in differentiation and pathogenicity in cell culture and in vivo. Parallel efforts enable targeted delivery of cytotoxic drugs or imaging agents. Third, RNA can be protected from serum nucleases by replacing the ribose 2'OH with 2'F, 2'O-methyl, 2'-amino or other chemical moieties. Current questions in this area are to understand how these modifications affect aptamer folding and the RNA-protein binding interface, and to adapt selection technologies to accelerate identification of nuclease-resistant aptamers. Finally, additional advances in selection technologies are being applied to enhance intracellular internalization and escape from endosome into the cytoplasm.