- BS in Microbiology and Cell Science. University of Florida (1999)
- PhD in Microbiology and Cell Science. University of Florida (2004)
- Postdoctoral Research Associate. University of Florida (2004)
My research in the Benner group has focused on the development of a novel technique (SNAP2) 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.
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) 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."
2'-Deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine
Kim, HJ; Leal, NA; Benner, SA
Bioorg. Med. Chem.
17 (10) 3728-3732 (2009)
2 '-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2 '-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2 '-deoxycytidine and 2 '-deoxyguanosine pair. (C) 2009 Elsevier Ltd. All rights reserved.
Synthetic Biology for Improved Personalized Medicine
Benner, SA; Hoshika, S; Sukeda, M; Hutter, D; Leal, NA; Yang, ZY; Chen, F
Nucleic Acids Symp. Ser.
52 (1) 243-244 (2008) doi: 10.1093/nass/nrn123
Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk.
Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis
Ruhland, A; Leal, N; Kima, PE
9 (1) 84-96 (2007)
Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NF kappa B and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NF kappa B signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.
PduL is an evolutionarily distinct phosphotransacylase involved in B-12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2
Liu, Y; Leal, NA; Sampson, EM; Johnson, CLV; Havemann, GD; Bobik, TA
189 (5) 1589-1596 (2007)
Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B-12-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V-max was 51.7 +/- 7.6 mu mol min(-1) mg(-1), and the K-m values for propionyl-PO42- and acetyl-PO42- were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.
In vivo expression of human ATP : cob(I)atamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
Erger, KE; Conlon, TJ; Leal, NA; Zori, R; Bobik, TA; Flotte, TR
J. Gene Med.
9 (6) 462-469 (2007)
Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B-12, adenosylcobalamin (AdoCbl). AdoCbl is. a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adenoassociated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/B16 mice at a dose of 1 x 10(10) and 1 x 10(11), particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken P-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments. Copyright (C) 2007 John Wiley & Sons, Ltd.
Dynamic assembly of primers on nucleic acid templates
Leal, NA; Sukeda, M; Benner, SA
Nucl. Acids Res.
34 4702-4710 (2006)
A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of 1 µM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.
(View all publications by Nicole Leal)