(provided by One can readily envision many applications for the unnatural base pairs applicant): The diversity of all living organisms is encoded within their DNA, where it is stably maintained through DNA replication, and then retrieved through transcription into RNA and translation into proteins. Thus, the diversity of life is limited by the four natural nucleotids that comprise DNA and RNA and the twenty natural amino acids that they encode. Drawn by the potential conceptual and practical ramifications, chemists and biologists have long been fascinated by the idea of expanding the genetic alphabet. This requires an unnatural base pair that is replicated and transcribed, both efficiently and with high fidelity and without any significant sequence bias. Expansion of the genetic alphabet would make possible the site-specific labeling of DNA and RNA - just as these biopolymers are receiving increased attention for applications ranging from novel materials to therapeutics. The ability to synthesize or evolve DNA/RNA that is site-specifically modified with functional groups of interest, and thus has activities outside the scope of their natural counterparts, promises to greatly increase their potential applications. Expansion of the genetic alphabet would also lay the foundation for the first semi-synthetic organism, able to store increased information in its DNA and retrieve it in th form of proteins with unnatural amino acids. In the previous funding period we developed the first unnatural base pair, d5SICS-dNaM, that is replicated and transcribed with efficiency and fidelity approaching that of a natural base pair and without any significant sequence bias. Molecular recognition within this pair is based not on complementary hydrogen- bonding, as with the natural base pairs, but on complementary hydrophobic and packing forces, more similar to the forces underlying protein structure and folding. We also developed a polymerase selection system to identify mutants that better recognize the unnatural base pair. With these tools in hand, we now propose to use a variety of synthetic and biological methods to: 1) Complete the characterization of the structural determinants of d5SICS-dNaM stability, replication, &transcription;2) Complete the optimization of (d)5SICS and (d)MMO2/(d)NaM for replication, transcription, and site-specific labeling of DNA and RNA;3) Demonstrate the utility of the expanded genetic alphabet for multiple in vitro applications;4) Optimize T7 phage DNA polymerases for the in vivo replication of DNA containing d5SICS-dNaM;and 5) Initiate efforts to expand the genetic alphabet in vivo by establishing a system in E. coli for the uptake of (d)NaMTP and (d)5SICSTP. The completion of these aims should elucidate the mechanisms underlying DNA replication, and make available general tools for the site-specific labeling of DNA and RNA, as well as tools for the evolution of polymerases with desired activities. Completion of the proposed work will also position us at the doorstep of creating the first semi-synthetic organism, able to store, retrieve, and evolve, with more information than natural organisms.

Public Health Relevance

The diversity of all living organisms is encoded within their DNA, where it is stably maintained through DNA replication, and then retrieved through transcription into RNA and translation into proteins. Our proposed research to expand the genetic alphabet promises to elucidate the mechanisms underlying DNA replication, and make available general tools for the site-specific labeling of DNA and RNA, as well as tools for the evolution of polymerases with desired activities. Completion of the proposed research will also position us at the doorstep of creating the first semi-synthetic organism, able to store, retrieve, and evolve, with more information than natural organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM060005-13S1
Application #
8895110
Study Section
Program Officer
Fabian, Miles
Project Start
1999-09-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
13
Fiscal Year
2014
Total Cost
$87,614
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Lavergne, Thomas; Lamichhane, Rajan; Malyshev, Denis A et al. (2016) FRET Characterization of Complex Conformational Changes in a Large 16S Ribosomal RNA Fragment Site-Specifically Labeled Using Unnatural Base Pairs. ACS Chem Biol 11:1347-53
Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia et al. (2016) The expanding world of DNA and RNA. Curr Opin Chem Biol 34:80-87
Adhikary, Ramkrishna; Yu, Wayne; Oda, Masayuki et al. (2015) Adaptive mutations alter antibody structure and dynamics during affinity maturation. Biochemistry 54:2085-93
Malyshev, Denis A; Romesberg, Floyd E (2015) The expanded genetic alphabet. Angew Chem Int Ed Engl 54:11930-44
Chen, Tingjian; Romesberg, Floyd E (2014) Directed polymerase evolution. FEBS Lett 588:219-29
Li, Lingjun; Degardin, Melissa; Lavergne, Thomas et al. (2014) Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. J Am Chem Soc 136:826-9
Dhami, Kirandeep; Malyshev, Denis A; Ordoukhanian, Phillip et al. (2014) Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet. Nucleic Acids Res 42:10235-44
Malyshev, Denis A; Dhami, Kirandeep; Lavergne, Thomas et al. (2014) A semi-synthetic organism with an expanded genetic alphabet. Nature 509:385-8
Lavergne, Thomas; Degardin, Melissa; Malyshev, Denis A et al. (2013) Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair. J Am Chem Soc 135:5408-19
Betz, Karin; Malyshev, Denis A; Lavergne, Thomas et al. (2013) Structural insights into DNA replication without hydrogen bonds. J Am Chem Soc 135:18637-43

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