Research

Cofactor-binding RNAs: Link to heading

In the last 35 years, many RNAs and DNAs have been discovered that bind the same cofactors proteins use to catalyze chemical reactions - like flavins, heme, cobalamins, and other small molecules. A surprising number of these cofactor-binding nucleic acids are good binders, but not very good catalysts. The best-characterized example we have of DNA and RNA binding a cofactor and catalyzing a chemical reaction is probably aptamers that bind to heme. These are surprisingly good peroxidase enzymes, and they can catalyze a lot of the same chemistry that the best-known protein peroxidases, like horseradish peroxidase, can catalyze. For many years, it was unclear if heme-nucleic acid complexes had any relevance to biology. Recent results from a few groups have shown that they could be.

We have shown that heme-nucleic acid complexes can exhibit some surprising behaviors in a few systems. In 2022, we found that a DNA duplex could switch back and forth between a catalytically active and a catalytically inactive form with heat, temperature, salt, and even vacuum as inputs. More recently, we observed that a RNA-heme complex could catalyze carbon-chlorine bond formation. To the best of our knowledge, this is the first time a ribozyme has done this!

We are working to discover new cofactor-binding RNAs and extend the capabilities of those we already have. This serves two goals - development of synthetic biological systems, and uncovering new roles for RNA in living systems.

Fluorescent RNAs: Link to heading

The study of biology would not look like it does today without genetically encoded fluorescent tags. Green fluorescent protein (GFP) and related proteins have proven phenomenally useful in this regard. They’re also an incredible gift from nature. The GFP we use is derived from a sequence originally found in the Aequorea victoria jellyfish. Remarkably, it’s not even green when it’s first synthesized by the ribosome - it’s colorless, like most proteins. To turn green (and fluorescent), it essentially performs surgery on itself, turning three amino acids into a fluorophore, with the rest of the protein providing a rigid pocket that activates the fluorescence of the green pigment. It does this without any help, too - a lot of proteins need cofactors or helper proteins, but not GFP.

We don’t yet know of a jellyfish for RNA, despite the fact that it’d be very useful if we did. That is, there’s not a natural fluorescent RNA. Over the last 10-15 years, lab-selected fluorescent RNAs that bind added pigments have proven to be powerful imaging and sensing tools along the lines of GFP. In some ways, they open up some unique opportunities - it is generally operationally more straightforward to make an RNA-based fluorescent sensor than a protein-based sensors. Conversely, there are some things that make them operationally a little harder to use, like the need to add a pigment.

In recent years, we’ve used fluorescent RNAs to monitor RNA folding in denaturing high-salt solution, detect pathogen RNA in a low-cost detection system (and again in a second-generation followup with even higher sensitivity), monitor nonenzymatic synthesis of RNA in a model prebiotic system, monitor outputs of a cell-free biocomputing system, monitor transfer of RNA and protein payloads between model synthetic cells, and generate and monitor “caged” redox-responsive RNAs that respond to the presence of hydrogen peroxide.

We are developing new fluorescent RNAs and finding new applications for existing ones.

How we do our science Link to heading

We use a wide range of tools to look at these systems. The core techniques for what we do are nucleic acid synthesis, purification, and handling, but everyone in the lab gets exposed to at least a few other techniques. Some that we’ve used in recent years are: in vitro selection, bioinformatics and machine learning, liposome preparation, biophysics, fluorescence microscopy, cytometry, CAD/3D printing/machining, embedded computing, tissue culture, NMR, cell-free transcription-translation (TXTL), and organic synthesis.