Dr. James Bashkin, D. Phil. - University of Missouri-St. Louis - Professor

Dr. James Bashkin

D. Phil.

University of Missouri-St. Louis

Professor

St. Louis, MO | United States

Main Specialties: Chemistry

Additional Specialties: Bioorganic chemistry, nucleic acids, HPV, antivirals

ORCID logohttps://orcid.org/0000-0001-6735-4733


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Dr. James Bashkin, D. Phil. - University of Missouri-St. Louis - Professor

Dr. James Bashkin

D. Phil.

Introduction

Primary Affiliation: University of Missouri-St. Louis - St. Louis, MO , United States

Specialties:

Additional Specialties:

Research Interests:


View Dr. James Bashkin’s Resume / CV

Education

Oct 1982
University of Oxford
D. Phil.
Organometallic Chemistry
Sep 1975 - Jul 1977
University of California Irvine
B.A.
Chemistry
Aug 1974
University of Arizona

Biology (one year)

Experience

Dec 2011
University of Missouri- St. Louis
Professor of Chemistry and Biochemistry
Inorganic, Organic and Biochemistry Divisions
Jun 1999 - Nov 2011
University of Missouri-St. Louis
Research Associate Professor
Chemistry and Biochemistry
May 2010
R01 Biophysics of Drug Interactions with Viral Episomes
PI
NIH NIAID 5 year grant
Oct 2003
NanoVir, LLC
Co-Founder and Director of Chemistry
Aug 2003
University of Missouri- St. Louis
Research Associate Professor
Department of Chemistry
May 1999
Monsanto Company
Research Advisor (Director Level 1)
Later Pharmacia, then Pfizer
May 1991
Washington University in St. Louis
Assistant Professor
Inorganic Chemistry
Aug 1985
Monsanto Corporate Research
Senior Research Chemist
Nov 1982
Harvard University
Postdoc and NIH Postdoc
Bioinorganic chemistry, to 5/85

Publications

30Publications

1400Reads

10Profile Views

1267PubMed Central Citations

DNA binding thermodynamics and site stoichiometry as a function of polyamide size.

Biochimie 2019 Jul 31;165:170-178. Epub 2019 Jul 31.

Department of Chemistry & Biochemistry, University of Missouri St. Louis, St. Louis, MO, 63121, USA. Electronic address:

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http://dx.doi.org/10.1016/j.biochi.2019.07.021DOI Listing
July 2019
3 Reads
2.963 Impact Factor

Thermodynamics and site stoichiometry of DNA binding by a large antiviral hairpin polyamide.

Biochimie 2019 Feb 24;157:149-157. Epub 2018 Nov 24.

Department of Chemistry & Biochemistry, University of Missouri St. Louis, St. Louis, MO, 63121, USA. Electronic address:

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https://linkinghub.elsevier.com/retrieve/pii/S03009084183033
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http://dx.doi.org/10.1016/j.biochi.2018.11.013DOI Listing
February 2019
199 Reads
2.963 Impact Factor

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

J Virol 2018 04 28;92(8). Epub 2018 Mar 28.

Department of Chemistry, Georgia State University, Atlanta, Georgia, USA

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

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http://dx.doi.org/10.1128/JVI.00146-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874401PMC
April 2018
270 Reads
3 Citations
4.439 Impact Factor

β-Alanine and N-terminal cationic substituents affect polyamide-DNA binding.

Org Biomol Chem 2017 Nov;15(46):9880-9888

Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as ?-alanine (?) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of ? and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the ?B motif. The ?B motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that ? inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a ? replacement between two imidazole heterocycles (Im?Im) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of ?s and cationic substituents.

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http://dx.doi.org/10.1039/c7ob02513kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763910PMC
November 2017
110 Reads
5 Citations
3.562 Impact Factor

What is the antiviral potential of pyrrole-imidazole polyamides?

Future Med Chem 2015 23;7(15):1953-5. Epub 2015 Oct 23.

Department of Chemistry & Biochemistry, Center for Nanoscience, University of Missouri-St Louis, 1 University Blvd, St Louis, MO 63121, USA.

A novel antiviral mechanism for polyamides is described after discussing prior work. N-Methylpyrrole (Py)- and N-methylimidazole (Im)-derived polyamides are higher homologs of natural product distamycin A, which binds sequence-selectively to AT-rich double-stranded DNA (dsDNA). Instead of interacting with DNA via Watson–Crick or Hoogsteen base pairing, polyamides recognize dsDNA by reading the ‘edges’ of intact Watson–Crick pairs in the minor groove of the double helix [1]. At sufficient concentrations, distamycin binds to dsDNA in a 2:1 ratio, antiparallel, and this arrangement has been mimicked with great success by longer, nominally linear polyamides that incorporate a flexible building block and bend near the middle, resembling hairpins. The hairpin motif is favored because it forms a double-stranded polyamide and allows the 2:1 style of binding to take place with a 1:1 molar ratio of (folded) polyamide to DNA. Aromatic rings stack in both the polyamide region and the DNA, which adds to hydrogen bonding, giving high affinity and a sequence specificity that can seem very high. A cationic guanidine or amidine group is found at the N- and/or C-termini of distamycin A and its natural analogs; this charge presumably improves both solubility and affinity to anionic DNA. These charged moieties are often replaced in synthetic polyamides by mono- and diamines, or more complex structures [2]. 

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http://dx.doi.org/10.4155/fmc.15.120DOI Listing
August 2016
9 Reads
7 Citations
4.000 Impact Factor

Interactions of two large antiviral polyamides with the long control region of HPV16.

Biochimie 2016 Aug 4;127:103-14. Epub 2016 May 4.

Department of Chemistry & Biochemistry and the Center for Nanoscience, University of Missouri St. Louis, St. Louis, MO 63121, USA. Electronic address:

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5'-W2GW7-3'; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM-20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5'-W2GW5GW4-3' via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicates a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

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http://dx.doi.org/10.1016/j.biochi.2016.04.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931995PMC
August 2016
62 Reads
8 Citations
2.963 Impact Factor

Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

Nucleic Acids Res 2015 Apr 30;43(8):4322-31. Epub 2015 Mar 30.

College of Pharmacy, Washington State University, Spokane, WA 99210, USA

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

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http://dx.doi.org/10.1093/nar/gkv267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417174PMC
April 2015
36 Reads
12 Citations
9.112 Impact Factor

Modulation of DNA-polyamide interaction by β-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

Org Biomol Chem 2014 Oct;12(38):7523-36

Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA.

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, ?-alanine (?), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that ? can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its ? derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double ?s. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The ? substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the ? substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

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http://dx.doi.org/10.1039/c4ob01456aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339220PMC
October 2014
14 Reads
17 Citations
3.562 Impact Factor

Binding studies of a large antiviral polyamide to a natural HPV sequence.

Biochimie 2014 Jul 26;102:83-91. Epub 2014 Feb 26.

Department of Chemistry & Biochemistry and the Center for Nanoscience, University of Missouri St. Louis, St. Louis, MO 63121, United States. Electronic address:

PA1 is a large hairpin polyamide (dImPyPy-?-PyPyPy-?-PyPy-?-PyPyPyPy-?-Ta; Py = pyrrole, Im = imidazole, ? = beta alanine) that targets the sequence 5'-WWGWWWWWWW-3' (W = A or T) and is effective in eliminating HPV16 in cell culture (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are its DNA binding properties toward a natural DNA, a 523 bp portion of HPV16 (2150-2672) containing three predicted perfect match sites. Strategies for obtaining binding data on large fragments using capillary electrophoresis are also described. Using an Fe EDTA conjugate of PA1, 19 affinity cleavage (AC) patterns were detected for this fragment. In many cases, there are multiple possible binding sequences (perfect, single and double mismatch sites) consistent with the AC data. Quantitative DNase I footprinting analysis indicates that perfect and most single mismatch sites bind PA1 with Kds between 0.7 and 4 nM, indicating excellent tolerance for the latter. Double mismatch sites exhibit Kds between 12 and 62 nM. A large fraction of the accessible sequence is susceptible to PA1 binding, much larger than predicted based on the literature of polyamide-DNA recognition rules.

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http://dx.doi.org/10.1016/j.biochi.2014.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047171PMC
July 2014
58 Reads
21 Citations
2.963 Impact Factor

DNA damage repair genes controlling human papillomavirus (HPV) episome levels under conditions of stability and extreme instability.

PLoS One 2013 2;8(10):e75406. Epub 2013 Oct 2.

NanoVir, Kalamazoo, Michigan, United States of America.

DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ?5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process.

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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075406PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788802PMC
June 2014
123 Reads
40 Citations
3.234 Impact Factor

DNA-Binding polyamides designed against E1, E2 binding sites of HPV DNA show dramatic anti-HPV activity in cell and tissue culture

Koeller KJ, Harris GD, Aston K, He G, Castaneda CH, Thornton MA, et al. DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents. Med Chem (Los Angeles). 2014;4:338-44. Epub 2014/05/20.

Med Chem (Los Angeles)

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of beta-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

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May 2014

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents.

Med Chem (Los Angeles) 2014 Feb;4:338-344

Department of Chemistry & Biochemistry, University of Missouri-St. Louis, St.Louis, MO 63121, USA ; NanoVir, LLC, Kalamazoo, MI 49008, USA.

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of ?-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2 International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

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http://dx.doi.org/10.4172/2161-0444.1000162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022477PMC
February 2014
69 Reads
18 Citations
1.910 Impact Factor

Different thermodynamic signatures for DNA minor groove binding with changes in salt concentration and temperature.

Chem Commun (Camb) 2013 Oct;49(76):8543-5

Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.

The effects of salt concentration and temperature on the thermodynamics of DNA minor groove binding have quite different signatures: binding enthalpy is salt concentration independent but temperature dependent. Conversely, binding free energy is salt dependent but essentially temperature independent through enthalpy-entropy compensation.

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http://dx.doi.org/10.1039/c3cc44569kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791883PMC
October 2013
14 Reads
11 Citations
6.834 Impact Factor

Mapping small DNA ligand hydroxyl radical footprinting and affinity cleavage products for capillary electrophoresis.

Anal Biochem 2013 Aug 19;439(2):99-101. Epub 2013 Apr 19.

Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri-St. Louis, St. Louis, MO 63121, USA.

The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence.

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http://dx.doi.org/10.1016/j.ab.2013.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713177PMC
August 2013
23 Reads
7 Citations
2.275 Impact Factor

Human papillomavirus episome stability is reduced by aphidicolin and controlled by DNA damage response pathways.

J Virol 2013 Apr 30;87(7):3979-89. Epub 2013 Jan 30.

NanoVir, Kalamazoo, Michigan, USA.

A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a little-understood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.

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http://dx.doi.org/10.1128/JVI.03473-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624211PMC
April 2013
13 Reads
50 Citations
4.439 Impact Factor

Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine.

Biochimie 2013 Feb 27;95(2):271-9. Epub 2012 Sep 27.

Department of Chemistry & Biochemistry, Center for Nanoscience, University of Missouri-St. Louis, One University Blvd., St. Louis, MO 63121, USA.

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ?-alanines as polyamide building blocks in place of N-methylpyrrole; ?-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ?-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ?-alanine to generate a ?/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ?-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

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http://dx.doi.org/10.1016/j.biochi.2012.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557744PMC
February 2013
69 Reads
18 Citations
2.963 Impact Factor

Correlation of local effects of DNA sequence and position of β-alanine inserts with polyamide-DNA complex binding affinities and kinetics.

Biochemistry 2012 Dec 28;51(49):9796-806. Epub 2012 Nov 28.

Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.

To improve our understanding of the effects of ?-alanine (?) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two ? derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of ? have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ?C(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that ? substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of ? substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.

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http://dx.doi.org/10.1021/bi301327vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567211PMC
December 2012
10 Reads
18 Citations
3.015 Impact Factor

Fluorescence assay of polyamide-DNA interactions.

Anal Biochem 2012 Apr 28;423(1):178-83. Epub 2012 Jan 28.

Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri-St. Louis, St. Louis, MO 63121, USA.

Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU near the PA binding site. In a study of 6 PAs, PA binding leads to a steady reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assay works equally well with both short (6- to 8-ring) and long (14-ring) PAs, and K(d) values ranging from approximately 1 nM to at least 140 nM were readily obtained using a simple monochromator or filter configuration. Competition assays provide a means to assessing possible dye interference, which can be negligible. The assay can also be used to determine PA extinction coefficients and to measure binding kinetics; thus, it is an accessible and versatile tool for the study of PA properties and PA-DNA interactions.

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http://dx.doi.org/10.1016/j.ab.2012.01.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299850PMC
April 2012
14 Reads
13 Citations
2.275 Impact Factor

HPV episome levels are potently decreased by pyrrole-imidazole polyamides.

Antiviral Res 2011 Aug 2;91(2):177-86. Epub 2011 Jun 2.

NanoVir, LLC, 4717 Campus Drive, Kalamazoo, MI 49008, USA.

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s?150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.

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http://dx.doi.org/10.1016/j.antiviral.2011.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135741PMC
August 2011
160 Reads
57 Citations
4.307 Impact Factor

Ion-exchange chromatography followed by ESI-MS for quantitative analysis of sugar monophosphates from glucose catabolism.

J Am Soc Mass Spectrom 2006 Jan 15;17(1):104-107. Epub 2005 Dec 15.

Department of Chemistry and Biochemistry, University of Missouri, St. Louis, Missouri, USA.

The aim of this work is to establish a quantitative method to determine the ratio of [U-(13)C] labeled to unlabeled hexose monophosphates isolated from yeast extracts. This is accomplished by anion exchange chromatography and mobile phase desalting followed by electrospray (ESI) mass spectrometry. We test the method with the analysis of a sample of biological origin. Previously developed analytical techniques are not adequate to accomplish mass spectrometric analysis of these and other small monosaccharide systems because of interference from salt clusters. By lowering the ionic strength of the mobile phase and using a simplified injection system to the mass spectrometer, we were able to obtain data on the relative abundance of the hexose monophosphates.

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http://link.springer.com/10.1016/j.jasms.2005.10.004
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http://dx.doi.org/10.1016/j.jasms.2005.10.004DOI Listing
January 2006
29 Reads
9 Citations
2.945 Impact Factor

Synthesis and evaluation of RNA transesterification efficiency using stereospecific serinol-terpyridine conjugates.

Nucleosides Nucleotides Nucleic Acids 2005 ;24(9):1309-23

Department of Chemistry, Washington University, St. Louis, Missouri, USA.

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http://dx.doi.org/10.1080/15257770500230426DOI Listing
December 2005
10 Reads
1.020 Impact Factor

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells.

Bioorg Med Chem Lett 2003 May;13(9):1565-70

Pharmacia Corporation, 700 Chesterfield Parkway North, Chesterfield, MO 63198, USA.

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.

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http://dx.doi.org/10.1016/s0960-894x(03)00152-5DOI Listing
May 2003
51 Reads
85 Citations
2.442 Impact Factor

Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA.

Nature 2002 Jun;417(6892):971-4

Bioscience Division/Structural Biology Center, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439, USA.

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http://dx.doi.org/10.1038/nature00833DOI Listing
June 2002
22 Reads
42.351 Impact Factor

Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1, 3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine

Bioconjugate Chem., 2001, 12 (6), pp 900–905 DOI: 10.1021/bc0100197

Bioconjugate chemistry

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)−serinol−terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic−RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5‘- and 3‘-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol−terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA−DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.

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November 2001

42 Citations

Impact Factor 4.818

11 Reads

Inorganic Mimics of Ribonucleases and Ribozymes: From Random Cleavage to Sequence-Specific Chemistry to Catalytic Antisense Drugs.

Chem Rev 1998 May;98(3):939-960

Department of Chemistry, Washington University, St. Louis, Missouri 63130.

Interest in functional mimics of ribozymes and ribonucleases is driven by a variety of scientific and medical goals. After a brief introduction, in which several working definitions, postulates, and premises are given, these goals are described and explained.  Metal-based strategies for RNA cleavage are then presented and contrasted with other approaches,  including biological and purely organic methods. The organic methods are exhaustively discussed in a companion article by Oivanen, Kuusela, and Lönnberg.  We describe the evolution of ribozyme mimics from early, nonspecific, ill-defined (but highly active) catalysts that were derived from “free metal ions in buffer”, to the advent of well-defined metal complexes that retained RNA cleavage activity.1,2  Such metal complexes allowed the central premise of this area to be tested and eventually led to proof of the concept that sequence-specific cleavage of RNA can be achieved by synthetic mimics. We explain how the continuing convergence of chemistry, biochemistry,  and molecular biology have helped shape research in this area, from the molecular design of early mimics to the choice of analytical methods for the screening of RNA transesterification catalysts. A  critical assessment of these analytical approaches is provided.  

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May 1998
10 Reads
388 Citations
45.661 Impact Factor

Introduction to RNA/DNA Cleavage.

Authors:
James K. Bashkin

Chem Rev 1998 May;98(3):937-938

Nucleic acids provide exciting and difficult challenges for chemists and biochemists. They are inherently important, yet are highly complex to study.  Many aspects of nucleic acid biochemistry are broadly  appreciated throughout the scientific community,  including the nature of the genetic code, the codon-anticodon relationships between mRNA and tRNA,  the importance of radiation damage to DNA,1 and the need for DNA repair. However, some very important processes are less widely recognized by nonspecialists.  For example, controlling the lifetimes of mRNA molecules is one of the means by which Nature governs protein synthesis.2 The antisense method for controlling gene expression, which has great potential for use in antiviral chemotherapy, uses a  similar idea: gene-specific deactivation or destruction of mRNA can inhibit the synthesis of harmful proteins.3-5 

With recent advances in nucleic acid synthesis,  analytical techniques, computational methods, and molecular biology procedures, it is becoming more and more feasible to study chemical reactions of DNA  and RNA from a highly detailed molecular perspective.  Work at the interface between nucleic acid  chemistry and biochemistry dates back many years,  and includes the pioneering studies of Dimroth,  Eichhorn, Lippard, Sigman, Dervan, and Barton.6-11  The gold standard for mechanistic chemical studies on reactions of DNA is provided by the work of  Stubbe and Kozarich.12,13 The chemical reactions that we focus on in this thematic issue are primarily cleavage reactions that split the biopolymer by attacking its backbone. The comprehensive reviews  presented here encompass enzymatic transesterification  and hydrolysis of biological phosphodiester  bonds, ribozyme-catalyzed reactions that occur in the  maturation of RNA, biomimetic hydrolysis and transesterification  of RNA by small organic and inorganic  molecules, photocleavage of DNA by natural and  synthetic reagents, photoprocesses of copper complexes  bound to DNA, oxidative cleavage via attack  on nucleobases and sugar moieties by either synthetic  reagents or natural products, and excision repair of damaged DNA. Both metal-dependent and metal independent nuclease enzymes are discussed. In such a rapidly advancing area, the forum provided by Chemical Reviews provides what we hope will be a timely and comprehensive view of these subjects.  Other valuable sources include a recent book on  nucleic acid chemistry14a and a related article on the  metal-dependent cleavage of DNA and RNA.14b

My intent has been to assemble a set of articles that will help propel DNA and RNA cleavage studies to the next frontiers, that will serve as a useful reference for some years to come, and that will aid the teaching of courses that address the interface between chemistry and biology. Some important areas of DNA and RNA cleavage were not addressed in this issue, including the enediynes, which have been extensively reviewed in recent years.15-18  I am grateful to all of the authors for their outstanding contributions and to the Editorial Staff of Chemical  Reviews and the ACS publications division for all of their help. I acknowledge NSF and the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support.

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May 1998
10 Reads
388 Citations
45.661 Impact Factor

Synthesis and Characterization of Cobalt-Cage Complexes with Pendant Phenol Groups.

Inorg Chem 1996 Jun;35(13):3828-3835

Department of Chemistry, Washington University, St. Louis, Missouri 63130-4899.

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http://dx.doi.org/10.1021/ic951610gDOI Listing
June 1996
12 Reads
4.762 Impact Factor

Top co-authors

Kevin J Koeller
Kevin J Koeller

University of Missouri St. Louis

8
Cynthia M Dupureur
Cynthia M Dupureur

University of Missouri-St. Louis

7
Karl Aston
Karl Aston

University of Missouri-St. Louis

7
Gaofei He
Gaofei He

University of Missouri-St. Louis

7
Shuo Wang
Shuo Wang

Beijing Tiantan Hospital

5
Terri G Edwards
Terri G Edwards

NanoVir

4
Chris Fisher
Chris Fisher

Weatherall Institute of Molecular Medicine

4
Jacquelyn Niederschulte
Jacquelyn Niederschulte

University of Missouri St. Louis

3
Elena Vasilieva
Elena Vasilieva

Moscow State University of Medicine and Dentistry

3
Yang Song
Yang Song

Key Laboratory of Soil Environment and Pollution Remediation

3