Research


Our laboratory is interested in unraveling the design principles that underlie the relationship between the sequence, structure and function of RNA molecules. Once thought to be a passive carrier of genetic information, RNAs are now understood to be essential to regulating and defending the genomes of all organisms. This broad array of function is hypothesized to be mediated by specific RNA structures that selectively interact with the cellular milieu. It is our goal to understand and design these structures so that we may utilize RNA function to engineer biomolecular systems as solutions to challenging problems in biology, medicine and biotechnology. To do this, we also work on developing technologies that can characterize RNA structures in massively high throughput to use as a diagnostic tool in our RNA engineering. This technology in turn opens new doors through which we can ask fundamental biological questions such as how specific RNA structures mediate cellular processes. With these fundamental investigations, we learn new RNA design principles that then feed back into our engineering methodology.

 

Pushing the Limits of RNA Design with Cellular Engineering

orthogA necessary first step in programming cells is developing large numbers of versatile, interoperable building blocks that can be combined together to create more complex function. RNA is an intriguing biomolecular substrate for this task because it has been shown to perform a wide array of function in biology, it is amenable to forward design, and there are cutting edge tools based on next generation sequencing that can now globally characterize its abundance, and soon its structures and interactions, across the entire cell. However, we still lack a complete set of basic design principles that we can use when designing RNAs to function independently with predictable function inside the cell.

One of our central interests is to change this by systematically uncovering and developing the principles with which we can engineer RNAs to perform essential functions necessary for programming cells. We are specifically interested in designing RNAs that can sense and integrate external and regulatory signals, regulate multiple aspects of gene expression, and propagate regulatory information. Shown here is one such example where we made the first steps to uncover the principles that underlie the specificity of interaction of an antisense RNA and its regulatory target, and showed how these regulators can be chained together to create the first reported RNA-mediated genetic network (link). We continue to push our ability to engineer RNA structure and function by asking fundamental engineering questions about how far we can go with RNA, and how we can build higher order genetic circuits out of our well-characterized RNA building blocks.

Related Papers:

  1. Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.
  2. Creating Small Transcription Activating RNAs, Nature Chemical Biology, 2015.
  3. A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future. Current Opinion in Chemical Biology, 2015.
  4. Characterizing and prototyping genetic networks with cell-free transcription-translation reactions, Methods, 2015.
  5. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies, Biotechnology and Bioengineering, 2015.
  6. Generating effective models and parameters for RNA genetic circuits, ACS Synthetic Biology, 2015.
  7. Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems, ACS SynBio, 2014.
  8. A modular strategy for engineering orthogonal chimeric RNA transcription regulators, NAR, 2013.
  9. The Centrality of RNA for Engineering Gene Expression, Biotechnology Journal, 2013 (Review).
  10. Versatile RNA-sensing transcriptional regulators for engineering genetic networks, PNAS, 2011.

 

Next Generation RNA Structure Characterization

In-cell_SHAPE-SeqWe lead the team that developed SHAPE-Seq, a technique that utilizes next generation sequencing to characterize RNA structures and interactions of complex mixtures of RNAs in a single experiment (link). As depicted, SHAPE-Seq utilizes advances in chemical probing to first modify RNAs in a structure dependent fashion. These RNAs are then converted into cDNAs by a process that is blocked by the modification. cDNAs are then sequenced, and sequencing reads are analyzed to uncover the locations of modifications on the RNA. This information is then used to infer structural features of the RNAs, or particular locations of interactions with other biomolecules.

We are currently developing improvements to SHAPE-Seq to be able to characterize vastly complex pools of RNA in a cellular context. SHAPE-Seq and its improvements are allowing us to systematically uncover the structural features that underlie specific RNA function, as well as be an integral tool in designing RNA sequences to specifically fold into structures and carry out designed function inside of the cell.

Related Papers:

  1. Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.
  2. Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Methods, 2016.
  3. RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs. Current Opinion in Biotechnology, 2016.
  4. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq. NAR, 2015.
  5. SHAPE-Seq 2.0: Systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next-generation sequencing. NAR, 2014.
  6. Multiplexed RNA structure characterization with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). PNAS, 2011.
  7. Modeling and automation of sequencing-based characterization of RNA structure. PNAS, 2011.
  8. RNA Structure Characterization from Chemical Mapping Experiments. Allerton Conference, 2011.

 

Harvesting RNA Design Principles from Nature

DesignThere has been a recent explosion in our understanding of the functional capability of RNAs. We are interested in studying specific natural RNA-mediated systems to uncover the basic principles in nature that give RNA this amazing array of function. Specifically, we are interested in the general principles by which RNAs interact with proteins so that we may use these to design specific interactions between our engineered RNA building blocks and other cellular components.