SPLIT DEOXYRIBOZYME SENSORS FOR NUCLEIC ACID ANALYSIS AND DIAGNOSTICS OF INFECTIOUS DISEASES
Nicole Aberdabbo,1 Rebecca Basch,1 Ryan Connelly,1 Carly Mitchell,2 Sheila Solarez1 and Yulia V. Gerasimova1
1Chemistry Department, University of Central Florida, Orlando FL 32816, USA
2Oviedo High School, Oviedo, FL 32765
Deoxyribozymes (Dz) are DNA molecules capable of catalyzing chemical reactions. Due to their structural versatility, biocompatibility, signal amplification ability and relatively low cost, Dz are widely used as scaffolds for biosensor design. Here we present a split Dz (sDz) approach for nucleic acid sensors, in which a Dz is divided into two subunits, which re-associate into the catalytically active construct only in the presence of a specific nucleic acid target. The target-inducible signal can be monitored for target detection and quantification. The advantages of sDz sensors include great selectivity of the target recognition down to a single base substitution in the target structure, as well tolerance to the secondary structure of the analyzed nucleic acid, which often represents a real challenge for conventional hybridization probes. Here, we present sDz sensors with fluorescent of colorimetric signal for highly selective recognition of bacterial and viral RNA, as well as amplified fragments of bacterial genome. The applications of the sensors for the RNA maturation monitoring and mutation analysis, as well as point-of-care compatible detection and genotyping of ZIKA virus and Mycobacterium tuberculosis are discussed.
Funding from the National Institutes of Health (NIH; 1R21AI123876-01A1) and the Florida Department of Health (7ZK33) is greatly appreciated.
Analyzing complex behavior of α7 nAChR binding sites
Alican Gulsevin1, Clare Stokes2, Roger L. Papke2, Nicole A. Horenstein1
1 - Department of Chemistry, University of Florida, Gainesville, FL, USA
2 - Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA
The α7 nicotinic acetylcholine receptor (nAChR) is an important ion channel involved in functions ranging from cognition and memory to inflammation. This receptor has a propensity to enter ligand induced desensitized states which are hypothesized to elicit anti-inflammatory effects. In many cases ion currents are not detectable, leading to the idea of metabotropic-like signaling from the receptor. In this study, computational chemistry and electrophysiology are used to explore the complex behavior of a group of molecules called silent agonists, that selectively place the α7 nAChR in a desensitized state that can be detected by co-application of positive allosteric modulators such as PNU-120596. Our findings suggest that ligands may bind the orthosteric binding domain with different “flipped” poses, and there is another possible binding site in the vestibule of the extracellular domain. The CRC and iCRC data generated for eight diethyl phenyl piperazines (DiEPPs) (partial agonists, silent agonists and an antagonist) on WT and Q57T receptor showed nearly half the potency relative to WT and a slight decrease in response. However, Q57T was able to nearly abolish silent agonist activity for DiEPP meta carboxamide. We discuss these results in the context of possible modes of action for silent agonists.
An anti-microbial peptide based intracellular delivery of nanocrystals
Anshika Kapur@, Scott Medina#, Wentao Wang@, Goutam Palui@, Xin Ji@, Joel P. Schneider# and Hedi Mattoussi@
@Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306, USA
# Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201
Engineered inorganic nanomaterials with tunable composition and controllable surface functionalization have emerged as potential imaging and diagnostic tools for integration with biological systems. However, little is known about the strategies to facilitate the movement of nanocrystals (NCs) across the cell membrane while circumventing endocytosis. We show that an anti-microbial peptide, SVS-1, can potentially be utilized to promote non-endocytic uptake of nanocrystals including luminescent quantum dots (QDs), metallic gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The N-terminal cysteine residue of the chemically synthesized peptide allows for covalent coupling to amine-functionalized NCs. Fluorescence imaging data show homogeneous distribution of the rapidly internalized NC-SVS-1 conjugates throughout the cytoplasm of different mammalian cell lines. The internalized NCs do not show any co-localization with labelled endosomes or nucleii. More importantly, additional endocytosis inhibition experiments imply that the conjugates are primarily internalized through physical translocation of the membrane rather than endocytosis. Live cell imaging data along with quantification by flow cytometry analysis also support our conclusions.
Structural and functional characterization of the colibactin resistance gene clbS
Prabhanshu Tripathi, Steven D. Bruner
Department of Chemistry, University of Florida, Gainesville, FL 32611, USA
Colibactin is an uncharacterized secondary metabolite produced by certain strains of E. coli present in the human gut. It induces double-stranded DNA breaks in eukaryotic cells promoting colorectal cancer and tumor formation under host inflammatory conditions. The colibactin biosynthetic pathway has been studied extensively by various groups since its discovery nearly a decade ago. The biosynthetic gene cluster consists of several NRPS, PKS, hybrid NRPS/PKS genes, a transporter and a resistance gene. The biosynthesis involves a prodrug-like resistance strategy where the inactive genotoxin is transported to the periplasm, cleaved and processed to the active form. Two genes clbM and clbQ from this pks island have already been structurally characterized in our laboratory. The ClbM structure was recently solved and was identified as a colibactin MATE transporter. Here, we report the X-ray structure of the colibactin resistance gene clbS, whose gene product protects the host bacteria from the genotoxic effects of colibactin. The structure was solved to 2.0 Å and reveals ClbS as a DNA-binding protein.
Sex determination of human remains using LAMP amplified Amelogenin gene and a deoxyribozyme sensor
Alexandra L. Smith
Dmitry M. Kolpashchikov
University of Central Florida
Sex identification of unknown remains is crucial to personal identification of human remains in anthropology and forensics. When conventional methods, such as metric or morphological analyses, are not an option due to the fragmented or prepubescent remains, molecular diagnostics are needed. The amelogenin gene, found on sex (X and Y) chromosomes, is the most common molecular marker used for sex determination because it exhibits sexual dimorphism in size and sequence. We develop a new method for fluorescent analysis of amelogenin gene for sex identification. In this assay, human DNA is amplified during a period of 15 min to 30 min by isothermal loop mediated amplification (LAMP) followed by analysis by binary deoxyribozyme sensors (BiDZ) for 60 min. High selectivity of the amelogenin sequences of X and Y chromosome was demonstrated. The assay promises to simplify molecular-based sex determination of human remains.
A Distributive ATP Grasp Ligase Macrolactonizes Multiple Microviridin Core Peptides within a Single Substrate
Yi Zhang,1 Kunhua Li,2 Guang Yang,1 Joshua L McBride,1 Steven D Bruner2 and Yousong Ding1
1Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, College of Pharmacy, University of Florida, Gainesville, Florida, 32610, USA.
2Department of Chemistry, University of Florida, Gainesville, Florida, 32611, USA.
Microviridins are a distinct type of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a cage-like architecture and are potent inhibitors of therapeutically-relevant serine proteases. Their biosynthesis requires the sequential installation of two ester and one amide linkages by two ATP grasp ligases. Functionally diverse enzymes contribute to astonishing chemical and functional richness of RiPPs and therefore offer opportunities to produce new analogs and to discover new enzymology. Of particular, enzymes that process multiple core peptides within a single precursor peptide are invaluable to investigate the combination of enzyme specificity and promiscuity. However, such enzymes have been known only in one example. Here we report in vitro characterization of an ATP grasp ligase AMdnC in a silent microviridin pathway of Anabaena sp. PCC7120, whose precursor peptide AMdnA contains three core peptides. AMdnC catalyzed an unprecedented multi-site macrolactonization on AMdnA. Its catalysis occurred in a distributive fashion and followed an unstrict N-to-C overall directionality. Furthermore, AMdnC processed engineered precursor peptides with one to four core peptides. Collectively, these results disclose the second example of enzyme with novel catalytic properties in processing multiple core peptides and suggest that highly organized synthetic biology modules exist in nature.