TSRI Training Program in Molecular Evolution

L.M. Walker, D.R. Burton,* et al.
The Scripps Research Institute,
IAVI Neutralizing Antibody Center
Science web publication (9/3/2009)
Chosen by M.G. Finn

Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1-infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1-infected individuals, primarily infected with non-clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from approximately 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses. 


H.H. Wang, F.J. Isaacs,* P.A. Carr, Z.Z. Sun, G. Xu, C.R. Forest, G.M. Church*. 
Harvard Medical School, Harvard University, MIT, and Georgia Tech
Nature, 460, 894-898(2009)
Chosen by Katie Petrie  
The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
M. Chandra, A. Sachdeva, S.K. Silverman*
University of Illinois at Urbana-Champaign
Nat. Chem. Biol.,  doi: 10.1038/nchembio.201 (2009)
Chosen by: Igor Rupniewski
Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and with a rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is noteworthy because many natural DNA nucleases are bimetallic protein enzymes. 
J. Grünewald, M.-L. Tsao, R. Perera, L. Dong, F. Niessen, B.G. Wen, D.M. Kubitz,
V.V. Smider, W. Ruf, M. Nasoff, R.A. Lerner, P.G. Schultz
The Scripps Research Institute and the Novartis Research Foundation. 
Proc. Natl. Acad. Sci. U.S.A.,105,11276-11280 (2008)
Chosen by M.G. Finn
The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-a) to p-nitrophenylaianine (pNO­2Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) -> Phe mutant. The antibodies generated against the pNO2Phe are highly cross-reactive with native mTNF-a and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.