- 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)
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.
Electrochemical Reduction and Oxidation of Eight Unnatural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode
Spacek, J., Karalkar, N., Fojta, M., Wang, J., Benner, S. A
, 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.
Hachimoji DNA and RNA: A genetic system with eight building blocks
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
(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.
The Challenge of Synthetic Biology. Synthetic Darwinism and the Aperiodic Crystal Structure.
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.
Tautomeric equilibria of iso-guanine and related purine
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.
Assays To Detect the Formation of Triphosphates of Unnatural
Nucleotides: Application to Escherichia coli Nucleoside Diphosphate
Mariko F. Matsuura, Ryan W. Shaw, Jennifer D. Moses, Hyo-Joong Kim, Myong-Jung Kim, Myong-Sang Kim, Shuichi Hoshika, Nilesh Karalkar, and Steven A. Benner
ACS Synthetic Biology
, American Chemical Society (2016) 5 (3), pp 234-240 DOI: 10.1021/acssynbio.5b00172
One frontier in synthetic biology seeks to move artificially
expanded genetic information systems (AEGIS) into natural living cells and to
arrange the metabolism of those cells to allow them to replicate plasmids built
from these unnatural genetic systems. In addition to requiring polymerases that
replicate AEGIS oligonucleotides, such cells require metabolic pathways that
biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those
polymerases. Such pathways generally require nucleoside and nucleotide kinases
to phosphorylate AEGIS nucleosides and nucleotides on the path to these
triphosphates. Thus, constructing such pathways focuses on engineering natural
nucleoside and nucleotide kinases, which often do not accept the unnatural
AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the
enzyme engineer to follow the kinase reaction, assays that are easily confused by
ATPase and other spurious activities that might arise through "site-directed
damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural
and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring
the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-
(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-
4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro
and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.
Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System
Karalkar NB, Leal NA, Kim MS, Bradley KM, Benner SA
ACS Synthetic Biology
, American Chemical Society (2016) doi: 10.1021/acssynbio.5b00276
As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ~92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ~86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.
Alternative Watson-Crick Synthetic
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.
Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups
Hutter, D; Kim, MJ; Karalkar, N; Leal, NA; Chen, F; Guggenheim, E; Visalakshi, V; Olejnik, J; Gordon, S; Benner, SA
Nuc. Nuc. Nuc. acids
29 (11) , Taylor & Francis Group 879-895 (2010)
Nucleoside triphosphates having a 3'-ONH(2) blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH(2) group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH(2) blocking group in "next generation sequencing."
Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations
Xu, YZ; Karalkar, NB; Kool, ET
19 (2) 148-152 (2001)
Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. the ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).
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