(7p) Engineering Functional Nucleic Acid Nano-Devices

“Single molecule RNA folding studied with optical trapping”, UC Berkeley

Advisors:: Ignacio Tinoco, Chemistry; Carlos Bustamante; Physics

Postdoctoral research:

“Shielded covalent probes for imaging and control of gene expression”, Caltech

Supervisor: Niles Pierce, Bioengineering

Research Interests:

Control of molecular structure and reactivity at the nanometer scale has been a dream of chemical engineers for years. For far longer, Nature has accomplished this feat with immense variation and scale, relying on bottom-up self-assembly of genetically evolved components to build structures and reaction pathways more complex than anything humans can design. Nucleic acid nanotechnology has emerged as an exciting field in which the programmable hybridization rules of base pairing are used to construct intricate two- and three-dimensional structures, carry out logic operations, and build molecular circuits and motors that operate at the smallest of scales. The primary theme of my proposed research is to leverage the advances of nucleic acid nanotechnology to build functional nano-devices capable of carrying out chemical transformations and probing biochemical systems in vitro and in vivo.

My graduate training focused on single-molecule studies of RNA folding, using optical trapping to measure the thermodynamics and kinetics of folding and unfolding reactions as a function of nucleic acid sequence, environmental conditions, and mechanical force. This provided a thorough grounding in the physical interactions that underlie nucleic acid self-assembly, as well as frontier areas such as non-equilibrium statistical mechanics and reaction pathway design.

My postdoctoral research aimed to apply our knowledge of nucleic acid thermodynamics and kinetics to the design and construction of dynamic, conformation-switching molecular nanodevices capable of performing chemical reactions with programmable targeting of biological molecules. I developed ‘shielded covalent probes’, nucleic acid hairpins that covalently capture target nucleic acid molecules with exquisite sensitivity and near-quantitative yield. These probes are currently being used in in situ imaging and gene expression, and were granted a US Patent.

My current research at the Institute for Molecular Engineering has expanded the applications of nucleic acid self-assembly to multiple length scales and novel chemistries. I have developed new methods for site-selective conjugation of nucleic acids to proteins and peptides, am investigating synthetic base pairing with metal ions for organometallic catalysis (in collaboration with Prof. Hosea Nelson at UCLA), and have led an extensive research program in polyelectrolyte assembly of nucleic acid – peptide nanoparticles with the laboratory of Prof. Matthew Tirrell. The common goal of these projects is applying our knowledge of nucleic acid interactions to produce useful molecular devices that operate on the nano-scale and interact with targets based on their specific genetic sequences, measuring and controlling biological outcomes with the utmost precision.

Future directions:

As a PI, I will apply my skills and training to engineer functional nucleic acid nanodevices that solve several pressing biochemical problems:

Programming chemical reactivity: Using the capabilities to form site-specific covalent linkages between oligonucleotides and proteins that I have developed, I plan to use nucleic acid scaffolds and nanopores to engineer enzymatic cascades in which the product of one reaction immediately becomes the substrate of another, avoiding the need for high intermediate concentrations or costly step-by-step separations. Additionally, I will continue a collaboration developing nucleic acids as conformationally-controllable ligands for organometallic catalysis; the ultimate goal of this project is to produce therapeutic molecules inside the specific cells that need them to treat disease.

Biomolecular phase transitions: In membraneless organelles and pathological aggregates, molecular conformation change drives phase separation in vivo. We recently found that the phase of nucleic acid-peptide complexes is controlled by nucleic acid hybridization. I plan to extend this work to full-length mRNA and proteins in order to determine the physical principles governing phase transitions in normal and pathological states. The same principles will enable the design of nanoparticles for more efficient delivery of therapeutic nucleic acids, a key challenge to realizing the potential of therapies such as siRNA.

Ribonucleoprotein engineering: Nature has evolved protein enzymes that operate with amazing effectiveness, but processing genetic information is primarily the job of nucleic acids, such as those at the core of natural ribonucleoproteins like the ribosome, spliceosome, and sequence specific nucleases like Dicer (RNAi) and CasX (CRISPR). By combining protein enzymes with the sequence recognition capabilities of nucleic acids, I hope to create synthetic ribonucleoproteins capable of therapeutic control of gene expression in vivo.

Teaching Interests:

I believe that teaching and research inform each other, and am excited to help students learn both foundational concepts and cutting-edge developments. As a graduate student, I had the opportunity to TA several courses in physics and chemistry, in both small and large format classes, including one introductory undergraduate class that was in the process of converting from traditional lecture-based to small group active learning. I completed a STEM pedagogy course as a graduate student, and have obtained a certificate from the Caltech Project in Effective Teaching as a postdoctoral researcher. Throughout my career, I have mentored undergraduate and graduate students, and look forward to doing so as a PI. As a faculty member, I am excited to teach students in core chemical engineering courses such as thermodynamics and chemical kinetics, and hope to develop an upper division/graduate course in biomolecular design.

Successful Proposals:

• NIH R01 (assisted, postdoctoral scholar)
• UCBREP Graduate Research and Education in Adaptive Biotechnology Training Grant (primary, graduate student)
• UChicago Institute for Translational Medicine Core Grant (primary, research scientist)

Selected Publications and Patents:

Journal Articles (12 published, 3 in preparation/review; h-index: 9)

• J.R. Vieregg and T-Y D. Tang, Polynucleotides in cellular mimics: coacervates and lipid vesicles.

Curr. Opin. Colloid & Interface Science, 26, 50-57 (2016). DOI: 10.1016/j.cocis.2016.09.004

• J.R. Vieregg, H.M. Nelson, B.M. Stoltz, and N.A. Pierce, Selective nucleic acid capture with shielded covalent probes.

J. Am. Chem Soc., 135, 9691-9699 (2013). DOI: 10.1021/ja4009216

• P.T.X. Li, J.R. Vieregg, I. Tinoco, Jr., How RNA unfolds and refolds.

Ann. Rev. Biochem. 77, 77-100 (2008). DOI: 10.1146/annurev.biochem.77.061206.174353

• J.R. Vieregg, W. Cheng, C. Bustamante, I. Tinoco, Jr. Measurement of the effect of monovalent cations on RNA hairpin stability.

J. Am. Chem. Soc. 129, 14966-73 (2007). DOI: 10.1021/ja074809o

Patents

US Patent # 8,658,780: Triggered Covalent Probes for Imaging and Silencing Gene Expression