- BS in Microbiology and Cell Science. University of Florida (1999)
- PhD in Microbiology and Cell Science. University of Florida (2004)
- Postdoctoral Research Associate. Microbiology and Cell Science, University of Florida (2004)
- Postdoctoral Research Associate. Department of Chemistry, University of Florida (2005)
- Associate Scientist at the Foundation for Applied Molecular Evolution, Alachua, FL (2006-current)
My research in the Benner group has focused on the development of a SNAP2, novel technique for the detection of specific DNA and/or RNA molecules in a biological mixture. This technique uses short oligonucleotide primers (6-8mers) that are complementary to a target sequence under conditions of dynamic equilibrium. The primers are modified such that an imine bond is formed when they are in close proximity, allowing for the discriminatory power of short oligonucleotide duplexes and an overall specificity of priming characteristic of longer oligonucleotides (14-16mers). In theory, these primers will only snap together, prime and extend in the presence of the target sequence. This technology can be applied towards the development of various DNA assays including the detection of single nucleotide changes within a target sequence.
I have also been working on the molecular aspects of reversibly terminated DNA sequencing. I have been involved in the development and optimization of Sequencing during Synthesis reactions (SdS) using reversible terminators. This technology can be applied to the development of a faster and less expensive method for genomic sequencing.
In the field of synthetic biology, I have been involved in developing the manipulative and analytical technology needed to support the conversion of six-letter information encoded in DNA to give the corresponding information in encoding RNA molecules in vitro. I have shown that T7 RNA polymerase and reverse transcriptase catalyze the transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, specifically Z and P. This work sets the stage for the next step in the development of an AEGIS synthetic biology, including the use of DNA containing extra codons based on the AEGIS expanded alphabet to encode mRNA and tRNA that might increase the number of amino acids within the protein lexicon.
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.
"Skinny" and "Fat" DNA: Two New Double Helices
Hoshika S, Singh I, Switzer C, Molt RW Jr, Leal NA, Kim MJ, Kim MS, Kim HJ, Georgiadis MM, Benner SA
J. Am. Chem. Soc.
(2018) Sep 19;140(37):11655-11660. doi: 10.1021/jacs.8b05042. Epub 2018 Sep 10
According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.
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.
Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components
Nicole A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan, and Steven A. Benner
ACS Synthetic Biology
, American Chemical Society (2015) Apr 17;4(4):407-13. doi: 10.1021/sb500268n
Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.
Ribonucleosides for an Artificially Expanded Genetic Information
Hyo-Joong Kim, Nicole A. Leal, Shuichi Hoshika, Steven A. Benner
J. Org. Chem.
(2014) 79 (7), pp 3194-3199
Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2?-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson?Crick complement,imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA.
Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns
Laos R, Shaw R, Leal NA, Gaucher E, Benner S.
(2013) 52, 5288-5294
Artificial genetic systems have been developed
by synthetic biologists over the past two decades to include
additional nucleotides that form additional nucleobase pairs
independent of the standard T:A and C:G pairs. Their use in
various tools to detect and analyze DNA and RNA requires
polymerases that synthesize duplex DNA containing unnatural
base pairs. This is especially true for nested polymerase chain
reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are
used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA
polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard
nucleotides, 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with
6-amino-5-nitro-3-(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed
evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template.
Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed
"heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change
in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the
template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the
general rule in directed evolution that "you get what you select for".
Conversion strategy using an expanded genetic alphabet to assay nucleic acids
Yang, Z., Durante, M., Glushakova, L., Sharma, N., Leal, N., Bradley, K., Chen, F., Benner, S. A.
Methods to detect DNA and RNA (collectively
xNA) are easily plagued by noise, false positives, and false
negatives, especially with increasing levels of multiplexing in
complex assay mixtures. Here, we describe assay architectures
that mitigate these problems by converting standard xNA
analyte sequences into sequences that incorporate nonstandard
nucleotides (Z and P). Z and P are extra DNA building blocks
that form tight nonstandard base pairs without cross-binding
to natural oligonucleotides containing G, A, C, and T
(GACT). The resulting improvements are assessed in an
assay that inverts the standard Luminex xTAG architecture,
placing a biotin on a primer (rather than on a triphosphate).
This primer is extended on the target to create a standard
GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a
polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that
is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces
higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures
improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of
competition from any natural oligonucleotides, even in complex biological samples.
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."
(View publication page for Nicole Leal)