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(386) 418-8085

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FfAME Our Team Nilesh Karalkar
Nilesh Karalkar

Senior Scientist

Nilesh Karalkar

  • (386) 418-8085

Research Summary

My current research involves chemically synthesized base pairs that will recognize each other through hydrogen bonds in a manner similar to natural bases (A,C,G,T) but with altered H-bonding directionality. This technology could be implemented in a wide variety of applications including mutation detection, mixed population genotyping, and multiplexed genetic analysis.

My past research has included the following topics:

  • Affinity purification of high value proteins. Since its discovery, affinity chromatography has evolved as one of the most powerful and effective fractionation techniques for the purification of proteins. The unique interaction between the target molecule and complementary ligand covalently attached to an insoluble matrix provides the specificity required for the isolation of biomolecules from complex mixtures, such as cell extracts. I have worked on developing a method/approach for the capture of molecules and/or assemblies that is mediated by interaction of a substrate with a capture phase.
  • Telomerase inhibitors. One of the enzymes found in 90 percent of cancer cells is a compound called telomerase; it replaces the bit of telomere clipped off after each cell division. If telomerase production can be turned on in normal cells, it seems reasonable that normal cells could become immortal. My past research has focused on inhibition of human telomerase (hTR). Towards this goal I have designed, synthesized, and evaluated novel lipid conjugated N3'-P5' oligonucleotide as telomerase inhibitors.
Research Focus:
  • Synthetic biology
  • Medicinal chemistry
  • Nucleic acids chemistry
  • DNA sequencing and synthesis
Education:
  • PhD in Chemistry. Mumbai University, India (1999)
  • Postdoctoral Research Associate. Standford University, CA (2001)
  • Research Scientist, Medicinal Chemistry. Geron Corporation, Menlo Park, CA (2003)
  • Scientist, Speciality Nucleic Acid Chemistry. Transgenomic, Boulder, CO (2006)

Publications

Spacek, J., Karalkar, N., Fojta, M., Wang, J., Benner, S. A Electrochimica acta, International Society of Electrochemistry (2020) 362:137210, DOI:10.1016/j.electacta.2020.137210

Recently we showed the reduction and oxidation of six natural 2'-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2'-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

Hoshika H, Leal N, Kim MJ, Kim MS, Karalkar NB, Kim HJ, Bates AM, Watkins Jr. NE, SantaLucia HA, Meyer AJ, DasGupta S, Piccirilli JA, Ellington AD, SantaLucia Jr. J, Georgiadis MM, Benner SA Science (2019) 22 Feb 2019: Vol. 363, Issue 6429, pp. 884-887. DOI: 10.1126/science.aat0971

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Karalkar, N.B. and Benner, S.A. Curr. Op. Chem Biol. (2018) 46C:188-195, DOI:10.1016/j.cbpa.2018.07.008

'Grand Challenges' offer ways to discover flaws in existing theory without first needing to guess what those flaws are. Our grand challenge here is to reproduce the Darwinism of terran biology, but on molecular platforms different from standard DNA. Access to Darwinism distinguishes the living from the non-living state. However, theory suggests that any biopolymer able to support Darwinism must (a) be able to form Schrödinger's 'aperiodic crystal', where different molecular components pack into a single crystal lattice, and (b) have a polyelectrolyte backbone. In 1953, the descriptive biology of Watson and Crick suggested DNA met Schrödinger's criertion, forming a linear crystal with geometrically similar building blocks supported on a polyelectrolye backbone. At the center of genetics were nucleobase pairs that fit into that crystal lattice by having both size complementarity and hydrogen bonding complementarity to enforce a constant geometry. This review covers experiments that show that by adhering to these two structural rules, the aperiodic crystal structure is maintained in DNA having 6 (or more) components. Further, this molecular system is shown to support Darwinism. Together with a deeper understanding of the role played in crystal formation by the poly-charged backbone and the intervening scaffolding, these results define how we might search for Darwinism, and therefore life, on Mars, Europa, Enceladus, and other watery lagoons in our Solar System.

Nilesh B. Karalkar, Kshitij Khare, Robert Molt, and Steven A. Benner Nuc. Nuc. Nuc. acids, Taylor & Francis Group (2017) Apr 3;36(4):256-274. doi: 10.1080/15257770.2016.1268694

Nucleobase pairs in DNA match hydrogen-bond donor and acceptor groups on the nucleobases. However, these can adopt more than one tautomeric form, and can consequently pair with nucleobases other than their canonical complements, possibly a source of natural mutation. These issues are now being revisited by synthetic biologists increasing the number of replicable pairs in DNA by exploiting unnatural hydrogen bonding patterns, where tautomerism can also create mutation. Here, we combine spectroscopic measurements on methylated analogs of isoguanine tautomers and tautomeric mixtures with statistical analyses to a set of isoguanine analogs, the complement of isocytosine, the 5th and 6th "letters" in DNA.

Steven A. Benner, Nilesh B. Karalkar, Shuichi Hoshika, Roberto Laos, Ryan W. Shaw, Mariko Matsuura, Diego Fajardo, and Patricia Moussatche Cold Spring Harb Perspect Biol, Cold Spring Harbor Laboratory Press (2016) doi: 10.1101/cshperspect.a023770

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
All My Publications