We are engineering bacterial, yeast, and mammalian cells with new unnatural 'parts' in order to provide them with additional function, that exceeds Nature's repertoire of biological processes. Specifically, we engineer cells with orthogonal biosynthetic pathways that enable them to site-specifically incorporate unnatural amino acids with new functions into proteins. Thus far, we have achieved the genetic encoding of amino acids that allow for protein bioconjugation, protein crosslinking, and light-activation of protein function. We applied the genetically encoded light-activated amino acids to the photochemical regulation of DNA polymerization, recombination, and transcription. Several of these projects are being conducted in collaboration with the Chin lab.

In addition, we are applying a more expedient approach of finding small molecules that modulate gene function in live animals, compared to the traditional drug discovery process. Here, we are interested in discovering small molecules that perturb or modulate developmental processes and pathways. In collaboration with Dr. Nascone-Yoder we are using the Xenopus frog embryo as a model organism, since it develops rapidly and can be easily exposed to synthetic organic compounds.

Traditional methods for studying gene functions in multicellular model organisms have several disadvantages, including the potential of lethal phenotypes due to a lack of temporal control. We are developing novel tools based on the interplay of small organic molecules and proteins, DNA, and RNA, which enable temporal and spatial control of gene function using light. These projects, together with our synthetic biology work (see above), are related to the field of optogenetics. By using light as an input signal that can be controlled with high precision, these methodologies will enable the elucidation of gene function with unprecedented resolution.

Recently, we identified a small molecule which phenocopies a common birth defect, heterotaxia, in these embryos and we are using this molecule for the investigation of the TGF-beta pathway involvement in this congenital disorder.

Several projects in the lab involve the discovery of small molecule inhibitors and activators of specific biological pathways that are implicated in human diseases, including cancer, HCV infection, and HIV infection. Besides representing novel chemical genetics tools for the investigation of those pathways, the discovered molecules also have significant potential as fundamentally new therapeutic agents.

The brief research overview below is only meant as an introduction to the ongoing chemical biology projects in the lab and is not comprehensive. More detailed information can be found in our publications. If you are interested in applying some of our methodologies in your research, feel free to request any materials.

We are developing synthetic approaches to a wide range of natural and unnatural molecules. The chemical synthesis of new compounds is the enabling technology for all biological discoveries and methdology developments in the lab.

In addition to chemical transformations, we are also investigating and applying enzymatic transformations of small organic molecules. In this context we have discovered substantial microwave effects in biocatalsysis, that will greatly enhance future enzymatic processes, which we are further investigating.

Over the past years, we successfully developed several synthetic routes to the monomeric building blocks of biological macromolecules. Our synthetic efforts led to the multistep assembly of light-activated phosphoramidites and unnatural amino acids. These molecules represent essential parts for our projects in photochemical genetics and synthetic biology (see below).

In addition, we are further developing the cyclotrimerization chemistry and are applying it to the synthesis of new ring structures, biological pathway modifiers, and new fluorophores that we are using in cellular labeling experiments.

We have developed microwave-mediated [2+2+2] cyclotrimerization reactions that enable the rapid construction of carbo- and heterocyclic molecules. We are applying this methodology to the assembly of several structures found in nature.  For example, we have been working on the synthesis of the natural products cryptoacetalide, citrinadine B, tylophorine, cannabinol, illudinine, and others.

We have also developed photochemical gene switches for pro- and eukaryotic cells. These switches are based on ribozymes and protein repressors in conjunction with small organic molecules that are modified with light-removable protecting groups. These genetic switches can be easily adapted to a wide range of applications, and we have already demonstrated a genetically encoded, light activated logic gate and bandpass filter.

Many signal transduction networks exist in mammalian cells. These networks are regulated with high spatial and temporal resolution and are involved in a wide range of cellular responses to external and internal stimuli. We are dissecting the circuitry of these networks using genetically encoded photocaged amino acids that enable the precise activation of specific network nodes at defined time points and locations.

Together with Dr. Jeff Yoder at NCSU, we are applying these photochemical gene switches to zebrafish embryos, which are excellent multicellular model organisms for the investigation of human biology. We already achieved the light-regulation of gene function with high temporal resolution using caged morpholinos in aquatic embryos.

Besides using synthetic biology approaches to the development of cellular light-switches, we are extensively applying our photocaged oligonucleotide chemistry to control gene function with high spatial and temporal resolution via antisense and DNA decoy approaches in mammalian tissue culture. This technology has proven to be very robust and applicable to a wide range of biological targets.

Another miRNA that we are targeting with small molecule inhibitors is miR-122. This miRNA is highly expressed in the liver and has been shown to be required for the replication of the hepatitis C virus. Moreover, it has recently been validated as a target for HCV therapeutics in primate and human clinical trials. The small molecule inhibitors discovered in our lab significantly reduce HCV levels in treated human liver cells.

Specifically, we are developing small molecules that target the microRNA pathway. MicroRNAs (miRNAs) are single-stranded noncoding RNAs of approximately 22 nucleotides that represent a new class of gene regulators. It is estimated that >30% of all genes and almost every genetic network are (in part) controlled by miRNAs. We discovered the first small molecule inhibitors of miRNAs, specifically miR-21. This miRNA is overexpressed in a wide range of cancers and we are currently using the small molecule as a probe to investigate miR-21 biogenesis. Moreover, in collaboration with Dr. Qihong Huang, we are evaluating the potential of our compounds as chemotherapeutic agents in a mouse model of brain cancer.