Maung Nyan Win

Higher-Order Cellular Information Processing with Synthetic RNA Devices

The engineering of biological systems is anticipated to provide effective solutions to challenges that include energy and food production, environmental quality, and health and medicine. Our ability to transmit information to and from living systems, and to process and act on information inside cells, is critical to advancing the scale and complexity at which we can engineer, manipulate, and probe biological systems. We developed a general approach for assembling RNA devices that can execute higher-order cellular information processing operations from standard components. The engineered devices can function as logic gates (AND, NOR, NAND, or OR gates) and signal filters, and exhibit cooperativity. RNA devices process and transmit molecular inputs to targeted protein outputs, linking computation to gene expression and thus the potential to control cellular function.

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Engineered biological systems hold promise in addressing pressing human needs in chemical processing, energy production, materials construction, and maintenance and enhancement of human health and the environment. However, significant advancements in our ability to engineer biological systems have been limited by the foundational tools available for reporting on, responding to, and controlling intracellular components in living systems. Portable and scalable platforms are needed for the reliable construction of such communication and control systems across diverse organisms. We report an extensible RNA-based framework for engineering ligand-controlled gene-regulatory systems, called ribozyme switches, that exhibits tunable regulation, design modularity, and target specificity. These switch platforms contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. We examined two modes of standardized information transmission between these domains and demonstrate a mechanism that allows for the reliable and modular assembly of functioning synthetic RNA switches and regulation of ribozyme activity in response to various effectors. In addition to demonstrating examples of small molecule-responsive, in vivo functional, allosteric hammerhead ribozymes, this work describes a general approach for the construction of portable and scalable gene-regulatory systems. We demonstrate the versatility of the platform in implementing application-specific control systems for small molecule-mediated regulation of cell growth and noninvasive in vivo sensing of metabolite production.

Frameworks for Programming Biological Function through RNA Parts and Devices

One of the long-term goals of synthetic biology is to reliably engineer biological systems that perform human-defined functions. Currently, researchers face several scientific and technical challenges in designing and building biological systems, one of which is associated with our limited ability to access, transmit, and control molecular information through the design of functional biomolecules exhibiting novel properties. The fields of RNA biology and nucleic acid engineering, along with the tremendous interdisciplinary growth of synthetic biology, are fueling advances in the emerging field of RNA programming in living systems. Researchers are designing functional RNA molecules that exhibit increasingly complex functions and integrating these molecules into cellular circuits to program higher-level biological functions. The continued integration and growth of RNA design and synthetic biology presents exciting potential to transform how we interact with and program biology.

RNA as a Versatile and Powerful Platform for Engineering Genetic Regulatory Tools

Abbreviations: RNA: ribonucleic acid, siRNA: small interfering RNA, miRNA: microRNA, mRNA: messenger RNA, ncRNA: non-coding RNA, DNA: deoxyribonucleic acid, UTR: untranslated region, RNAi: RNA interference, GC: guanine and cytosine, RBS: ribosomal-binding site, SD: Shine-Dalgarno, TPP: thiamine pyrophosphate, SELEX: Systematic Evolution of Ligands by EXponential enrichment, PCR: polymerase chain reaction, GUC: guanine, uridine, and cytosine, ATP: adenosine triphosphate, FMN: flavin mononucleotide, GlcN6P: glucosamine-6-phosphate, PTGS: post-transcriptional gene silencing, dsRNA: double-stranded RNA, RISC: RNAi-induced silencing complex, pre-miRNA: precursor hairpin transcript encoding microRNA, pri-miRNA: primary hairpin transcript encoding microRNA, RNP: ribonucleoprotein, miRNP: microribonucleoprotein, pol III: RNA polymerase III, shRNA: short hairpin RNA, pol II: RNA polymerase II, VEGF: vesicular endothelial growth factor, ELISA: enzyme-linked immunosorbent assay, HIV-1: human immunodeficiency virus 1, SPR: surface plasmon resonance, FRET: fluorescence resonance energy transfer, HCV: hepatitis C virus, NS3: non-structural protein 3, CTLA-4: cytotoxic T lymphocyte antigen-4, PSMA: prostate-specific membrane antigen. Biotechnology and Genetic Engineering Reviews Vol. 24, 311-346 (2007)

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines.

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270μg/L, 61μg/L, and 3700μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.