Arkady Mustaev

New Jersey Institute of Technology, Newark, New Jersey, United States

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Publications (58)460.41 Total impact

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    ABSTRACT: Recent X-ray crystallographic studies of Pol II in complex with the general transcription factor (GTF) IIB have begun to provide insights into the mechanism of transcription initiation. These structures have also shed light on the architecture of the transcription preinitiation complex (PIC). However, structural characterization of a functional PIC is still lacking, and even the topological arrangement of the GTFs in the Pol II complex is a matter of contention. We have extended our activity-based affinity crosslinking studies, initially developed to investigate the interaction of bacterial RNA polymerase with σ, to the eukaryotic transcription machinery. Towards that end, we sought to identify GTFs that are within the Pol II active site in a functioning PIC. We provide biochemical evidence that TFIIB is located within ∼9 Å of the -2 site of promoter DNA, where it is positioned to play a role in de novo transcription initiation.
    PLoS ONE 03/2015; 10(3):e0119007. DOI:10.1371/journal.pone.0119007 · 3.53 Impact Factor
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    ABSTRACT: Widespread fluoroquinolone resistance has drawn attention to quinazolinediones (diones), fluoroquinolone-like topoisomerase poisons that are unaffected by common quinolone-resistance mutations. To better understand differences between quinolones and diones, we examined their impact on the formation of cleaved complexes (drug-topoisomerase-DNA complexes in which the DNA moiety is broken) with gyrase, one of two bacterial targets of the drugs. Formation of cleaved complexes, measured by linearization of a circular DNA substrate, required lower concentrations of quinolone than dione. The reverse reaction, detected as resealing of DNA breaks in cleaved complexes, required higher temperatures and EDTA concentrations for quinolones than diones. The greater stability of quinolone-containing complexes was attributed to the unique ability of the quinolone C3/C4 keto acid to complex with magnesium and form a previously-described drug-magnesium-water bridge with GyrA-Ser83 and GyrA-Asp87. A nearby substitution in GyrA (G81C) reduced activity differences between quinolone and dione, indicating that resistance due to this variation derives from perturbation of the magnesium-water bridge. To increase dione activity, we examined a relatively small, flexible C-7-3-(aminomethyl)pyrrolidinyl substituent, which is distal to the bridging C3/C4 keto acid substituent of quinolones. The 3-(aminomethyl)pyrrolidinyl group at position C-7 was capable of forming binding interactions with GyrB-Glu466, as indicated by inspection of crystal structures, computer-aided docking, and measurement of cleaved-complex formation with mutant and wild-type GyrB proteins. Thus, modification of dione C-7 substituents constitutes a strategy for obtaining compounds active against common quinolone-resistant mutants.
    ACS Chemical Biology 10/2014; 9(12). DOI:10.1021/cb500629k · 5.36 Impact Factor
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    ABSTRACT: Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT-rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.
    Journal of Molecular Biology 10/2014; 426(24). DOI:10.1016/j.jmb.2014.10.005 · 3.96 Impact Factor
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    ABSTRACT: DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes due to the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from X-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed crosslinked cleaved complexes with mutant GyrB-466Cys gyrase, as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, crosslinking was also readily seen with complexes formed by mutant GyrA-81Cys gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The crosslink between fluoroquinolone and GyrA-81Cys gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-81Cys variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-89Cys). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug crosslink. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Since the GyrA-81 and GyrB-466 residues are far apart (17 A) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.
    Journal of Biological Chemistry 02/2014; 289(18). DOI:10.1074/jbc.M113.529164 · 4.60 Impact Factor
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    ABSTRACT: The aim of this study was to test the growth inhibition activity of isothiocyanates (ITC), defense compounds of plants, against common human microbial pathogens. In this study we have tested the growth inhibitory activity of a diverse collection of new and previously known representative ITC of various structural classes against pathogenic bacteria, fungi and molds by a serial dilution method. Generally, the compounds were more active against Gram-positive bacteria and fungi exhibiting species-specific bacteriostatic or bactericidal effect. The most active compounds inhibited the growth of both drug-susceptible and multi drug resistant (MDR) pathogens at micromolar concentrations. In the case of Mycobacterium tuberculosis some compounds were more active against MDR, rather than against susceptible strains. The average anti-microbial activity for some of new derivatives was significantly higher than previously reported for the most active ITC compounds. The structure-activity relationship (SAR) established for various classes of ITC with Bacillus cereus (model organism for B. anthracis) followed a distinct pattern, thereby enabling prediction of new more efficient inhibitors. Remarkably, tested bacteria failed to develop resistance to ITC. While effectively inhibiting microbial growth, ITCs displayed moderate toxicity towards eukaryotic cells. High antimicrobial activity coupled with moderate toxicity grants further thorough studies of the ITC compounds aimed at elucidation of their cellular targets and inhibitory mechanism. This systematic study identified new ITC compounds highly active against common human microbial pathogens at the concentrations comparable with those for currently used antimicrobial drugs (e.g. rifampicin, fluconazole). Tested representative pathogens do not develop resistance to the inhibitors. These properties justify further evaluation of ITC compounds as potential antimicrobial agents for medicinal use and for industrial applications. This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 06/2013; DOI:10.1111/jam.12288 · 2.39 Impact Factor
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    ABSTRACT: Rifampicin (Rif) is powerful broad spectrum antibiotic that targets bacterial RNA polymerase (RNAP) by blocking the transcript exit channel. The performance of the drug can be further enhanced by tagging with active chemical groups that produce collateral damage. We explored this principle by tethering Rif to Fe2+-EDTA chelate. Modified drug retained high binding affinity to RNAP and caused localized cleavage of the enzyme and promoter DNA. Analysis of the degradation products revealed the cleavage of RNAP β subunit at the sites involved in the drug binding, while DNA was selectively seized in the vicinity of the transcription start site. The synthesized Rif derivative exemplifies "aggressive" types of drugs that can be especially useful for TB treatment by attacking the nongrowing dormant form of the mycobacterium, which is hardly susceptible to "passive" drugs.
    Bioconjugate Chemistry 03/2013; 24(3). DOI:10.1021/bc3005667 · 4.82 Impact Factor
  • Shyamala Pillai, Lev Krasnoperov, Arkady Mustaev
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    ABSTRACT: Lanthanide ions luminescence has long life time enabling highly sensitive detection in time-gated mode. The synthesis of reactive lanthanide probes for covalent labeling of the objects of interest is cumbersome task due to the large size of the probes, complex multi-step procedures and the presence of sensitive groups, which often prevents introduction of reactive cross-linking functions optimal for conjugation. We suggest simple synthetic protocol for luminescent europium chelates based on serendipitous reaction yielding acylating compounds, whose reactivity is comparable to that of commonly used N-hydroxysuccinimide (NHS) esters. The probes react with proteins at pH 7.0 within several minutes at ambient temperature displaying high coupling efficiency. The resulting conjugates survive electrophoretic separation under denaturing conditions, which makes the labels useful in proteomic studies that rely on high detection sensitivity.
    Journal of Photochemistry and Photobiology A Chemistry 03/2013; 255:16-23. DOI:10.1016/j.jphotochem.2013.01.007 · 2.29 Impact Factor
  • Laura Wirpsza, Lev Krasnoperov, Arkady Mustaev
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    ABSTRACT: Luminescent lanthanide ion complexes are distinguished by unique light emitting properties that enable both highly sensitive detection of lanthanide labels attached to biomolecules and contrast imaging of various micro objects (cells, nanoparticles, etc.). Previously, we synthesized amine-reactive cs124-based luminescent lanthanide chelates with improved brightness and metal retention. Here we report the synthesis of new thiol-reactive derivatives of the same compounds including bromoacetamido-, and maleimido- forms of cs124 and cs124CF3 fluorophores. Maleimido-compounds displayed exceptional reactivity instantaneously coupling to thiols at physiological conditions at micromolar probes concentrations. Surprisingly, they displayed strong quenching by adjacent maleimido-group, which was completely eliminated after reaction with thiols, thereby enabling their simple detection by monitoring the light emission of the reaction mixture. This reaction can be used for hyper-sensitive determination of biologically important sulphydryl compounds (e.g. glutathione, co-enzyme A, etc.) in time-resolved mode.
    Journal of Photochemistry and Photobiology A Chemistry 02/2013; 251(1):30-37. DOI:10.1016/j.jphotochem.2012.12.008 · 2.29 Impact Factor
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    ABSTRACT: Precise transcription by cellular RNA polymerase requires efficient removal of onn-cognate nucleotide residues occasionally incorporated. Mis-incorporation causes transcription elongation complex (TEC) to backtrack, releasing single strand 3 prime RNA segment bearing non-cognate residue, which is hydrolyzed by the active center that carries two Mg2+ ions. However, in most X-ray structures only one Mg2+ is present. This Mg2+ is tightly bound to the active center aspartates, creating inactive stable state. The first residue of single strand RNA segment in backtracked TEC strongly promotes transcript hydrolytic cleavage by establishing a network of interactions that force a shift of stably bound Mg2+ to release some of its aspartate coordination valences for binding to the second Mg2+ enabling catalysis. Such rearrangement that we call active center tuning (ACT) occurs when all recognition contacts of active center-bound RNA segment are established and verified by tolerance to stress. Transcription factor Gre builds on ACT mechanism in the same reaction by increasing the retention of the second Mg2+ and by activating the attacking water, causing 3000-4000 fold reaction acceleration and strongly reinforcing proofreading. Unified mechanism for RNA synthesis and degradation by RNAP predicts that ACT also executes NTP selection thereby contributing to high transcription fidelity.
    Journal of Biological Chemistry 01/2013; 288(9). DOI:10.1074/jbc.M112.424002 · 4.60 Impact Factor
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    ABSTRACT: Lanthanide ion luminescence has a long lifetime enabling highly sensitive detection in time-gated mode. The sensitivity can be further increased by using multiple luminescent labels attached to a carrier molecule, which can be conjugated to an object of interest. We found that up to 30 lanthanide chelates can be attached to avidin creating highly bright constructs. These constructs with Eu(3+) chelates display synergistic effect that enhance the brightness of heavily modified samples, while the opposite effect was observed for Tb(3+) chelates thereby significantly reducing their light emission. This undesirable quenching of Tb(3+) luminophores was completely suppressed by the introduction of an aromatic spacer between the chelate and the protein attachment site. The estimated detection limit for the conjugates is in the 10(-14)-10(-15) M range. We demonstrated a high sensitivity for the new probes by using them to label living cells of bacterial and mammalian origin.
    Journal of photochemistry and photobiology. B, Biology 11/2012; 116:22-9. DOI:10.1016/j.jphotobiol.2012.07.001 · 2.80 Impact Factor
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    ABSTRACT: The diagnosis of invasive fungal infections from radiographic imaging is non-specific and problematic. As a first step toward increasing specificity, we describe the development of a broad-spectrum fungal-specific targeting molecule, which when modified with a fluorescent label fully retains its targeting properties, and provides a basis for future imaging applications.
    Medical mycology: official publication of the International Society for Human and Animal Mycology 05/2012; DOI:10.3109/13693786.2012.685767 · 2.26 Impact Factor
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    ABSTRACT: A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers—compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy. Electronic supplementary material The online version of this article (doi:10.1007/s10895-012-1039-z) contains supplementary material, which is available to authorized users.
    Journal of Fluorescence 03/2012; 22(4):1021-32. DOI:10.1007/s10895-012-1039-z · 1.67 Impact Factor
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    ABSTRACT: New luminescent lanthanide chelates containing thiol-, amine-, and click-reactive groups in antenna-fluorophore moieties were synthesized. The chelates include diethylenetriaminepentaacetic acid (DTPA) coupled to two types of chromophores: 7-amino-4-trifluoromethyl-2(1H) quinolinone, and 7-amino-4-trifluoromethyl-2-alkoxyquinoline. The synthesized compounds were characterized using NMR, light absorption, steady-state and time-resolved fluorescent spectroscopy. Some of the compounds displayed high brightness with Tb3+, Eu3+, and Dy3+. Obtained reactive lanthanide chelates can be easily attached to biological molecules. The probes demonstrated high performance in molecular beaconbased DNA hybridization assays (sub-pico molar detection limit), in bacterial proteome labeling, and in live cell imaging.
    Proceedings of SPIE - The International Society for Optical Engineering 01/2012; 8233(82331C):1-12. DOI:10.1117/12.921056 · 0.20 Impact Factor
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    ABSTRACT: Abstract-Luminescent lanthanide probes possess long emission lifetime, which enables their hypersensitive detection in time-resolved mode that avoids short-lived background fluorescence. Sharply spiked emission spectra of lanthanide probes and large Stokes shifts further enhance detection sensitivity, which is about 1000 fold higher than that for regular fluorescent probes. The wide spread of this promising technology is limited by high cost of commercially available compounds, which is mainly due to their complex structure and laborious synthetic procedure. In this study, new efficient strategies to simplify the synthesis of the probes were explored. New lanthanide chelates, containing click-, and amine-reactive cross-linking groups, which are highly bright and can be produced with high yield, are synthesized and characterized.
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    ABSTRACT: Novel fluoroquinolone derivatives substituted with a 2-thioalkyl moiety, with and without a concomitant 3-carboxylate group, were synthesized to evaluate the effect of C-2 thioalkyl substituents on gyrase binding and inhibition. The presence of a 2-thioalkyl group universally decreased activity as compared to parent fluoroquinolones. However, with derivatives of moxifloxacin the presence of either a 2-thioalkyl group or a 3-carboxylate moiety increased activity over the 2,3-unsubstituted derivative. Energy minimization of structures provides an explanation for relative activities of fluoroquinolones having a C-2 thio moiety.
    Bioorganic & medicinal chemistry letters 06/2011; 21(15):4585-8. DOI:10.1016/j.bmcl.2011.05.112 · 2.33 Impact Factor
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    ABSTRACT: Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the MIC for wild-type cells, were lower. Addition of a 3-amino group to the 2,4-dione core increased relative activity against mutants, while alteration of the 8-methoxy group to a methyl or of the 2,4-dione core to a 1,3-dione core lowered activity against mutants. A GyrA G89C bacterial variant was strikingly susceptible to most of the diones tested; in contrast, low susceptibility to fluoroquinolones was observed. Many of the bacteriostatic differences between diones and fluoroquinolones were explained by interactions at the N terminus of GyrA helix IV revealed by recently published X-ray structures of drug-topoisomerase-DNA complexes. When lethal activity was normalized to the MIC in order to minimize the effects of drug uptake, efflux, and ternary complex formation, a 3-amino-2,4-dione exhibited killing activity comparable to that of a cognate fluoroquinolone. Surprisingly, the lethal activity of the dione was inhibited less by chloramphenicol than that of the cognate fluoroquinolone. This observation adds the 2,4-dione structural motif to the list of structural features known to impart lethality to fluoroquinolone-like compounds in the absence of protein synthesis, a phenomenon that is not explained by X-ray structures of drug-enzyme-DNA complexes.
    Antimicrobial Agents and Chemotherapy 03/2011; 55(5):2335-43. DOI:10.1128/AAC.00033-11 · 4.45 Impact Factor
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    ABSTRACT: A variety of contemporary anal. platforms in tech. and biol. applications take advantage of labeling the objects of interest with fluorescent or luminescent tracers. Luminescent tracers take advantage of the unique property of some lanthanide metals to absorb and emit light. Long life time of lanthanide emission allows temporal gating of the signal, which avoids the short-lived background of interfering sample components. This property in combination with large Stokes shift contributes to extreme sensitivity of detection (ca. 10-13-10-14 M), which makes lanthanide-based probes suitable for large variety of challenging tasks ( e.g. Intracellular detection of single DNA/RNA, or protein mols., microbial pathogen detection in human specimens, tracing anal., etc.). Luminescent probes include antenna fluorophore that absorbs the light and transfers the excitation energy to a lanthanide tethered to antenna through chelation. The probe also contains crosslinking group that allows covalent labeling of the mol. of interest. Despite great potentials of lanthanide-based tracers, the wide spread of the technol. is impeded by very high price of com. available probes, which is due to their complex structure. The goal of our research was development of new strategies for the synthesis of lanthanide probes. In the course of this work we synthesized new antena-fluorophores, developed new methods for introduction of the crosslinking groups in the luminescent probes and elucidated the mechanisms of the chem. reactions leading to principal synthetic intermediates. We also synthesized new quinoline-based fluorophores with useful fluorescent properties and coupled their derivs. to DNA oligonucleotides using click chem. to obtain sensitive hybridization probes. Synthesized compds. were characterized using NMR, steady-state and time-resolved fluorescence spectroscopy, as well a UV absorption spectroscopy. [on SciFinder(R)]
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    ABSTRACT: The invention relates to novel luminescent compns. of matter contg. a fluorophore, synthetic methods for making the compns., macromol. conjugates of the compns., and the use of the compns. in various methods of detection. The invention also provides kits contg. the compns. and their conjugates for use in the methods of detection. DTPA-cs124-CF3-NCS (probe 1) (I) (prepn. given) was chelated with europium ion (from europium trichloride). Oligonucleotide 5'-amino-CTTCGTCCACAAACACAACTCCTGAAG-3'-Blackhole Quencher 2 was labeled with europium-I and used to detect a complementary DNA target. Escherichia coli treated with a biotin deriv. was labeled with streptavidin modified with multiple luminescent probes. [on SciFinder(R)]
    Year: 01/2011
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    ABSTRACT: Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.
    Bioconjugate Chemistry 02/2010; 21(2):319-27. DOI:10.1021/bc900403n · 4.82 Impact Factor
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    ABSTRACT: Chelate complexes of four lanthanide ions (Eu3+, Tb3+, Dy3+, and Sm3+) were studied to det. luminescence lifetimes and spectra characterization when excited by excimer laser radiation at 351 nm. Luminescence collected by a photomultiplier tube mounted on a spectrograph averaged was to det. the luminescence lifetimes in deionized regular (H2O) and heavy water (D2O). Lifetimes ranged from microseconds for probes with Sm3+ and Dy3+ ions and milliseconds for Eu3+ and Tb3+ derivs. Luminescence spectra acquired used a gated ICCD camera coupled to the spectrograph. Synthesized compds. were used in mol. beacon hybridization probes to assess their validity for biol. applications. Time-gated mode of detection avoided short-lived background fluorescence of the media providing high sensitivity, which was better than 1 pM. This concn. is about 50-100 times lower than conventional fluorescence-based mol. beacons and 10-60 times better than previously reported lanthanide-based hybridization probes. [on SciFinder(R)]

Publication Stats

3k Citations
460.41 Total Impact Points

Institutions

  • 2015
    • New Jersey Institute of Technology
      Newark, New Jersey, United States
  • 2009–2014
    • Rutgers New Jersey Medical School
      • • Department of Microbiology and Molecular Genetics
      • • Public Health Research Institute
      • • Department of Medicine
      Newark, New Jersey, United States
  • 2011
    • University of Iowa
      • College of Pharmacy
      Iowa City, Iowa, United States
  • 1993–2009
    • Public Health research Institute, India
      Davis, California, United States
  • 2002–2006
    • Russian Academy of Sciences
      • Institute of Molecular Genetics
      Moscow, Moscow, Russia
  • 1998–2001
    • New York University
      New York City, New York, United States
  • 1992
    • CUNY Graduate Center
      New York City, New York, United States