Research

 
 

Foundational Training in Organic Chemistry

As an undergraduate at Columbia College, Virginia received foundational training in organic chemistry.  Among others, she took undergraduate- and graduate-level organic chemistry courses with Professors George Flynn, Thomas Katz, and Ronald Breslow.  She carried out laboratory research with Professor Breslow where she synthetized a photoaffinity label that ultimately was part of a much larger effort to develop a first-in-class histone deacetylase inhibitor.  She graduated with a B.A. in Biochemistry summa cum laude.  To this day she enjoys teaching undergraduate Organic Chemistry and advising the undergraduate Biochemistry Majors.  Virginia was recognized with a Columbia College John Jay Award in 2005.  

Y. Webb, X. Zhou, L. Ngo, V.W. Cornish, J. Stahl, H. Erdjument-Bromage, P. Tempst, R. Rifkind, P. Marks, R. Breslow and V. Richon. "Photoaffinity Labeling and Mass Spectrometry Identify Ribosomal Protein S3 as a Potential Target for Hybrid Polar Cytodifferentiation Agents." J. Biol. Chem., 274, 14280-14287 (1999). HTML | PDF

Early Days of Chemical Biology

As a Ph.D. student in Chemistry at the University of California at Berkeley, Virginia was privileged to work in the early days of chemical biology on the unnatural amino acid mutagenesis project in Professor Peter Schultz’s laboratory. She worked on the technology in the early 1990s, when the unnatural amino acyl-tRNAs were prepared by chemoenzymatic synthesis and then incorporated site-specifically into proteins in an E. coli S30 cell-free extract. To advance the technology she applied it to questions in protein stability and the incorporation of biophysical probes. In her final publication with Professor Schultz in 1996, they reported the incorporation of a “ketone handle” for orthogonal introduction of biophysical probes and other molecules via post-translational modification of an unnatural amino acid with a ketone side chain. 

V.W. Cornish, D. Mendel, P.G. Schultz. "Probing Protein Structure and Function with an Expanded Genetic Code." Angew. Chem. Int. Ed. Engl., 34, 621-633 (1995). (Invited Review). HTML | PDF

V.W. Cornish, K.M. Hahn, P.G. Schultz. "Site-Specific Protein Modification Using a Ketone Handle." J. Am. Chem. Soc.,118, 8150-8151 (1996). HTML | PDF

Pioneering Independent Research in Yeast Synthetic Biology

Virginia is the Helena Rubinstein Professor in the Department of Chemistry and a Founding member of the Department of Systems Biology at Columbia University. Beginning as a postdoctoral fellow at in the Department of Biology at MIT under the mentorship of Professor Robert Sauer in the late 1990s, her laboratory has carried out pioneering work in synthetic biology in the yeast Saccharomyces cerevisiae over the past 25 years. Her initial contribution to the field, a project Professor Sauer allowed her to initiate independently during her post-doc at MIT and that she continued after assuming her independent position at Columbia, was pioneering the idea that genetic selections can be adapted for the in vivo directed evolution of chemistry not natural to the cell, which they coined “chemical complementation.” First, her laboratory developed the chemical complementation system for in vivo directed evolution of enzymes. Her laboratory then further developed in vivo technologies for DNA library assembly and mutagenesis for directed evolution inside of cells. In addition, as a spin-off of chemical complementation, her laboratory developed the TMP-tag as a chemical surrogate to the fluorescent proteins for selectively labeling proteins with small molecule fluorophores and other probes inside the cell. Finally, building off her Ph.D. research with Professor Schultz, her laboratory contributed to the field of unnatural amino acid incorporation by the translational machinery. About a decade ago her laboratory began to wind down both chemical biology projects (live cell imaging, unnatural amino acid mutagenesis), focus their research on their yeast synthetic biology work, and begin translating the yeast synthetic biology tools to applications in human health applications.  They began by developing a living yeast biosensor that can serve as a diagnostic in the short term and be incorporated into sense-respond yeast therapeutic communities in the longer term. They reported a scalable yeast communication language. Finally they began efforts to make living yeast therapeutics. Yeast engineered with synthetic biology is poised to be a transformative therapeutic platform, and they are excited to develop and translate yeast communities with sense-respond and other higher order functions over the next decade.

Independent Research in Chemical Biology

  • In collaboration with Professor Michael Sheetz in a project initiated by Professor Larry Miller when he was a post-doctoral fellow in Virginia’s laboratory, the Cornish laboratory developed the TMP-tag as a chemical surrogate to the fluorescent proteins. In the TMP-tag, rather than labeling a protein by fusing it at the genetic level with a fluorescent protein, the protein is fused to E. coli dihydrofolate reductase (eDHFR) and then labeled inside a living cell with cell-permeable trimethoprim (TMP)-fluorophore or other molecules. While the fluorescent proteins are the tools of choice for more straightforward applications, their chromophore is difficult to engineer because it is formed by a rearrangement of amino acids in the -barrel of the protein. With the TMP-tag and other chemical tags, the advantage is that the fluorophore/biophysical probe is independent of the labeling reaction and so can be designed to have higher photon outputs and other specialized properties. In collaboration, the Cornish laboratory developed non-covalent TMP-tags, covalent TMP-tags, TMP-tags for single-molecule imaging, TMP-tags for super-resolution imaging, and other applications. The TMP-tag is widely used for live cell imaging, and the Cornish laboratory used it in collaboration to study the focal adhesion complex and the spliceosome at single-molecule resolution. The Cornish laboratory developed related technologies for multi-“color” imaging on ~100 resolvable “colors”. Finally, the high affinity interaction between TMP and eDHFR has been utilized broadly in the field of chemical biology not only for live cell imaging but also for applications in chemical biology including controlled protein degradation, activation, post-translational modification, cellular localization, and other modalities.

    L. Miller, J. Sable, P. Goelet, M. Sheetz, V.W. Cornish. "Methotrexate Conjugates: A Molecular In Vivo Protein Tag." Angew. Chem., 116, 1704-1707 (2004); Angew. Chem. Int. Ed., 43, 1672-1675 (2004). HTML | PDF

    L. Miller, Y. Cai, M. Sheetz, V.W. Cornish. "In Vivo Labeling with Trimethoprim Conjugates: A Flexible Chemical Tag." Nature Methods, 2, 255-257 (2005). HTML | PDF

    R. Wombacher, M. Heidbreder, S. van de Linde, M.P. Sheetz, M. Heilemann*, V.W. Cornish*, M. Sauer*. “Live Cell Super-Resolution Imaging with Trimethoprim Conjugates.” Nature Methods, 7, 717-719 (2010). HTML | PDF

    A.A. Hoskins, L.J. Friedman, S.S. Gallagher, D.J. Crawford, E.G. Anderson, R. Wombacher, N. Ramirez, V.W. Cornish, J. Gelles*, M.J. Moore*. “Ordered and Dynamic Assembly of Single Spliceosomes.” Science, 331, 1289-1295 (2011). HTML | PDF

    A. Anzalone, Z. Chen, T. Wang, V.W. Cornish. “A Common Diaryl Ether Intermediate for the Gram-Scale Synthesis of Oxazine and Xanthene Fluorophores.” Angew. Chem., 52, 650-654 (2013). HTML | PDF

    L. Wei, Z. Chen, L. Shi, R. Long, A.V. Anzalone, L. Zhang, F. Hu, R. Yuste, V.W. Cornish, W. Min. “Super-Multiplex Vibrational Imaging.”  Nature, 544, 465-470 (2017). HTML | PDF

    A.V. Anzalone*, M. Jimenez*, V.W. Cornish*.  “FRAME-tags: a scalable palette of genetically encoded ratiometric fluorescent barcodes for multiplexed live cell tracking.”  bioRxiv, 04.09.436507 (2021). PDF

    X. Hu, C. Jing, X. Xu, N. Nakazawa, V.W. Cornish, F.M. Margadant, M.P. Sheetz. “Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy.” Nano Lett., 16, 4062-4068 (2016). HTML | PDF

     M.A. Shandell, J.R. Quejada, M. Yazawa, V.W. Cornish, R.S. Kass.  “Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP.”  Biochemistry, 117, 1352-1363 (2019). HTML | PDF

  • In her independent laboratory in collaboration, Dr. Tony Forster, Professor Steve Blacklow, her former Ph.D. student Zhongping Tan and Virginia demonstrated for the first time the translation of a defined unnatural oligomer reassigning multiple sense codons in a row using a purified translation system and chemoenzymatically synthesized aminoacyl-tRNAs. They published several manuscripts building on this initial publication working towards the goal of synthesis of oligomers other than the natural polypeptides by the translational machinery. This had been a long-standing challenge in the field and has opened up an entire sub-field in the unnatural amino acid community. Subsequently, in collaboration with Professors Ruben Gonzalez and Tom Leyh, her laboratory developed an experimental framework for discovering where in the elongation cycle of translation in E. coli unnatural amino acids are discriminated by the translational machinery. This work resulted in a series of manuscripts detailing the surprising finding that D-amino acids can be incorporated by the translational machinery but are discriminated in the P-site of the ribosome likely by engaging peptide stalling mechanisms. This forward-looking mechanistic work ultimately will be important for efforts to engineer the translational machinery for the synthesis of unnatural oligomers and the incorporation of building blocks that further diverge from the natural amino acids. Finally, in collaboration with Professor Rocky Kass, former Ph.D. student Mia Shandell used a fluorescent amino acid to study the mechanism of channel gating in a Voltage-Gated Sodium Channel. Virginia still considers unnatural amino acid mutagenesis as one of the unique tools she brings to the field of synthetic biology because of her background in the technology and in organic and protein chemistry.

    A. Forster, Z. Tan, M.N.L. Nalam, H. Lin, H. Qu, V.W. Cornish, S. Blacklow. "Programming Peptidomimetic Syntheses by Translating Genetic Codes Designed De Novo." Proc. Natl. Acad. Sci. USA, 100, 6353-6357 (2003). Featured in Chem. Biol., 10, 586-587 (2003) and in Chem. & Eng. News, 82, 64-68 (2004). HTML | PDF

    M.T. Englander, J.L. Avins, R.C. Fleisher, B. Liu, P.R. Effraim, J. Wang, K. Schulten, T.S. Leyh, R.L. Gonzalez Jr.*, V.W. Cornish*. “The Ribosome Can Discriminate the Chirality of Amino Acids Within Its Peptidyl Transferase Center.” Proc. Natl. Acad. Sci. USA, 112, 6038-6039 (2015). HTML | PDF

    M.A. Shandell, J.R. Quejada, M. Yazawa, V.W. Cornish, R.S. Kass. “Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP.” Biochemistry, 117, 1352-1363 (2019).

Independent Research in Yeast Synthetic Biology

  • Chemical complementation is a project that Virginia independently initiated during her post-doc at MIT and today is the foundation of her research at Columbia. This project put forward the idea that genetic selections can be adapted for the directed evolution of chemistry not natural to the cell. Chemical complementation is a high-throughput assay for enzyme activity that is reaction independent. This assay is based on a small-molecule yeast 3-hybrid system where a chemical inducer of dimerization (CID) bridges the two halves of a transcription factor. In chemical complementation, enzyme catalysis of bond formation or cleavage is detected as covalent coupling of the CID linker to control dimerization of the transcription factor and hence transcription of a reporter gene in vivo. We developed the selection using cephem hydrolysis by a cephalosporinase as a proof-of-principle. We demonstrated the generality of the selection by applying it to the directed evolution of glycosynthase and cellulase enzymes.

    H. Lin, W. Abida, R.T. Sauer, V.W. Cornish. “Dexamethasone-Methotrexate: An Efficient Chemical Inducer of Protein Dimerization In Vivo.” J. Am. Chem. Soc., 122, 4247-4248 (2000). DOI: 10.1021/ja9941532

    K. Baker, et al. "Chemical Complementation: A Reaction-Independent, High-Throughput Genetic Assay for Enzyme Catalysis." Proc. Natl. Acad. Sci. USA, 99, 16537-16542 (2002). PMCID: PMC139179.

    H. Lin, H. Tao, V.W. Cornish. "Directed Evolution of a Glycosynthase Via Chemical Complementation." J. Am. Chem. Soc., 126, 15051-15059 (2004). PMID: 15548001

    P. Peralta-Yahya, et al. “A High-Throughput Selection for Cellulase Catalysts Using Chemical Complementation.” J. Am. Chem. Soc., 130, 17446-17452 (2008). PMCID: PMC2981063

  • The Chemical Complementation project in our laboratory creating high-throughput screens and selections for enzyme catalysis spurred our interest in developing high throughput methods for DNA library assembly and mutagenesis. Our laboratory developed a yeast-based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co-transformed with single-stranded mutagenic oligonucleotides. This system was shown to select for functional variants from a library of 106 variants. Our laboratory has also developed the robust method “Reiterative Recombination” for building multigene pathways directly in the yeast chromosome. Lastly, our laboratory developed a technology we call “Heritable Recombination” in which a library cassette plasmid enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in S. cerevisiae. Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 1013.

    N. Pirakitikulr, N. Ostrov, P. Peralta-Yahya, V.W. Cornish. “PCRless Library Mutagenesis via Oligonucleotide Recombination in Yeast.” Protein Sci., 19, 2336-2346 (2010). PMCID: PMC3009401

    D.W. Romanini, P. Peralta-Yahya, V. Mondol, V.W. Cornish. "A Heritable Recombination System for Synthetic Darwinian Evolution in Yeast." ACS Synth. Biol., 1, 602–609 (2012). PMCID: PMC3569010

    L.M. Wingler, V.W. Cornish. “Reiterative Recombination for the In Vivo Assembly of Libraries of Multi- Gene Pathways.” Proc. Natl. Acad. Sci. USA, 108, 15135-15140 (2011). PMCID: PMC3174608

  • Building on our 2017 Science Advances proof-of-principle publication of a living yeast biosensor for fungal pathogens, we are using synthetic biology to engineer living yeast biosensors with a modular G-protein coupled receptor (GPCR) that recognizes an analyte, a signal transduction pathway, and finally a read-out detectable by the naked eye. The GPCR can rapidly be engineered to recognize diverse analytes using directed evolution. Endogenous yeast GPCR signaling pathways couple activation of the GPCR to transcription of a visual read-out such as lycopene, the pigment that turns tomatoes and other fruits red. The diagnostic can be readily scaled by fermentation, distributed at room-temperature in dried form, and used by non-experts at-home with no specialized reagents or equipment.