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Dmitry Kolpashchikov
Associate Research Scientist
650 W168th St, BB806, New York, NY 10032
Phone: (212)342-5610
Fax: (212)305-3475
Email:dk2111@columbia.edu

Academic Qualification

PhD

Training

2002 – 2003 –  Postdoctoral Research Scientist, Columbia University, New York, USA
2000 – 2002 –  Postdoctoral Fellow, National Institute of Genetics, Mishima, Japan
1999 – Ph.D. Bioorganic Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Russia
1994 – M.S. Chemistry, Novosibirsk State University, Russia.

Discipiline

Bio-organic Chemistry

Research Interests

• Bioorganic chemistry and biochemistry of nucleic acids
• Nucleic acid-based biosensors and logic circuits
• Selective recognition of proteins and nucleic acids
• Structural DNA nanotechnology
• Biomolecular engineering
• Synthetic biology
• Nucleic acid analysis
• Protein-nucleic acid interactions
• Structural and dynamic aspects of protein-ligand interactions
• Approaches for inactivation and cross-linking of proteins and nucleic acids
  

Honors And Awards

• Young Talented Scientist Award of Siberian Branch of Russian Academy of Science. 2000–2003
• Postdoctoral Fellowship from Centre of Excellence (COE) program of the Ministry of Education, Culture, Sport,  Science, and Technology of Japan. 2001–2002
• Postdoctoral Fellowship from Centre of Excellence (COE) program of the Ministry of Education, Culture, Sport, Science, and Technology of Japan. 2000–2001

Research Group

Our research is focused on binary approach for highly selective biopolymer recognition. The general idea behind this approach is as follows. A biopolymer (a protein or a nucleic acid) contains two ligand binding sites in close proximity. The two ligand analogs are conjugated to functional groups that are able to form a detectable signal, when specifically bound to the biopolymer. In our studies this signal is either a chemical reaction or an increase in fluorescence.

Principal scheme of a binary probe: two ligands are bound to the adjacent sties of a biopolymer and form a detectable signal.

Extramural grant support

2006-2008: NIH Exploratory/Developmental Research Grant Program "Nucleic Acid Analysis Using Deoxyribozyme Technology" Grant # 1 R21 HG004060. Principal Investigator

Current Projects

• Deoxyribozyme technology for nucleic acid analysis. This project aims to deliver a new PCR-free technique for nucleic acid analysis based on deoxyribozymes, which is cheap, rapid, and simple.
• Binary affinity reagents for highly selective inactivation of biopolymers. This project aims to develop new antiviral drugs based of covalent cross-linking of viral proteins and nucleic acids.
• Logic circuits made of DNA. The aim of this project is to establish a basis for creation of a DNA computer. In particular, two major complications of molecular circuit design will be solved: (i) Integration of molecular gates, and (ii) organization of the gates in arrays on a 2D platform.

Recent Results

One example of binary probe for nucleic acid analysis is binary malachite green aptamer (Figure 1). The probe consists of malachite green – a triphenylmethane dye - and two short RNA strands each of which comprises a fragment complementary to the analyte molecule (DNA binding arms) and a fragment of a malachite green aptamer (MGA).  The two RNA strands form MGA upon hybridization to the adjacent positions of the nucleic acid analyte. The resultant tertiary complex is able to bind malachite green and enhance the fluorescence of the dye, thus monitoring the presence of the nucleic acid in solution.  The probe reliably discriminated against 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at room temperature in near physiological buffer (Kolpashchikov D.M. (2005) JACS). 

Figure 1. Binary malachite green aptamer.

Second embodiment of binary approach is binary DNA probe (Figure 2). This probe combines binary approach with structural constrains and is even more selective than Malachite Green aptamer probe. Binary DNA probe consists of two DNA stands (A and B) and a moclecular beacon (MB). Both strand A and B comprise the fragments complementary to MB (MB binding arms) and the fragments complementary to the nucleic acid analyte (analyte binding arms). Each analyte binding arm contains a structural constraint in the form of a pentanucleotide stem. The analyte binding arm and the MB1 binding arm are connected through triethylene glycole linkers. In the absence of a nucleic acid analyte the strands are unbound in solution; MB is in the form of a hairpin (Figure 2 left) and the fluorescence signal is low. Addition of A20 DNA analyte triggers the formation of a quaternary complex (Figure 2, right). The fluorophore (FAM) is remote from the quencher (Dabcyl) in this complex, which results in high fluorescence. The extremely high selectivity of the probe is predetermined by cooperative hybridization of the two relatively short (10 nucleotide) DNA hairpin fragments to the analyte. Binary DNA probe fluorescently reports the presence of 0.5% of the analyte in excess amount of a single base substituted oligodeoxyribonucleotide and distinguishes single nucleotide substitutions at any position of a 20-mer oligonucleotide at room temperature (Kolpashchikov D.M. (2006) JACS).

Figure 2. Binary DNA probe.

Binary deoxyribozyme probe (Figure 3) allows not only highly selective target recognition but also improves detection sensitivity by catalytic amplification of the positive signals. Deoxyribozyme E6, obtained earlier by Breaker and Joyce, was split into two halves, and the analyte recognition arms were added to each half forming a binary deoxyribozyme, biE6. In the presence of a DNA analty the two subunits of the probe hybridize to the abutting fragments of the analyte and re-form the catalytic core. The active enzyme cleaved the fluorophore and quencher labeled reporter substrate and increased the fluorescence of the solution. The probe recognized single nucleotide substitutions in 20-mer oligodeoxyribonucleotide at room temperature and detected the analyte at the concentrations above 1 nM (Kolpashchikov D.M. (2007) ChemBioChem).

Figure 3. Binary deoxyribozyme probe

Publications

• Kolpashchikov D.M. (2007) A Binary Deoxyribozyme for Nucleic Acid Analysis. ChemBioChem 8, 2039-2042.
• Kolpashchikov D.M. (2006) Binary DNA Probe for Highly Specific Nucleic Acid Recognition. Journal of the American Chemical Society, 128, 10625-10628.
• Kolpashchikov D.M. (2005) Binary Malachite Green Aptamer for Fluorescent Detection of Nucleic Acids. Journal of the American Chemical Society, 127, 12442-12443.
• Kolpashchikov D.M., Stojanovic M.N. (2005) Boolean Control of Aptameric Binding States. Journal of the American Chemical Society, 127, 11348-11341.
• Stojanovic M.N., Semova S., Kolpashchikov D., Macdonald J., Morgan C., Stefanovic D. (2005) Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. Journal of the American Chemical Society, 127, 6914-6915.
• Stojanovic M.N., Kolpashchikov D.M. (2004) Modular aptameric sensors. Journal of the American Chemical Society, 126, 9266-9270.
• Kolpashchikov D.M, Honda A., Ishihama A.  (2004) Structure-Function Relationships of Influenza Virus RNA Polymerase: Primer-binding Site on PB1 Subunit. Biochemistry, 43, 5882-5887.
• Kolpashchikov D.M. (2003) Superaffinity Labeling of Proteins: Approaches and Techniques. J. Biomol. Struct. Dyn., 21, 55-64.
• Dezhurov S.V., Khodyreva S.N., Rechkunova N.I., Kolpashchikov D.M., Lavrik O.I. (2003) A Comparative study of the modification efficiency of DNA polymerase and DNA template by the DNA primers with various photoreactive group at their 3’-termini. Russian Journal of Bioorganic Chemistry, 29, 75-82.
• Lebedeva N.A., Kolpashchikov D.M., Rechkunova N.I., Khodyreva S.N., Lavrik O.I. (2002) Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents. Biochemistry (Moscow), 67, 807-814.
• Lavrik O.I., Kolpashchikov D.M., Prasad R., Sobol R.W., Wilson S.H. (2002) Binary system for selective photoaffinity labeling of base excision repair DNA polymerases. Nucleic Acids Research, 30, e73.
• Zakharenko A.L., Kolpashchikov D.M., Khodyreva S.N., Lavrik O.I., Menendez-Arias L. (2001) Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP. Biochemistry (Moscow), 66, 999-1007.
• Lebedeva N.A., Kolpashchikov D.M., Rechkunova N.I., Khodyreva S.N., Lavrik O.I. (2001) A binary system of photoreagents for high-efficiency labeling of DNA polymerases. Biochemical and Biophysical Research Communications, 287, 530-535.
• Kolpashchikov D.M., Hughes P., Favre A., Baldacci G., Lavrik O.I. (2001) Localization of the large subunit of replication factor C near the 5' end of DNA primers. Journal of Molecular Recognition, 14, 239-244.
• Kolpashchikov D.M., Khodyreva S.N., Khlimankov D.Y., Wold M.S., Favre A., Lavrik O.I. (2001) Polarity of human replication protein A binding to DNA. Nucleic Acids Research, 29.  373-379.
• Kolpashchikov D.M., Khodyreva S.N., Lavrik O.I. (2000) Replicative protein A exhibits a specific polarity in binding single-stranded DNA. Doklady Akademii Nauk, 372, 824-826.
• Kolpashchikov D.M., Ivanova T.M., Bogachev V.S., Nasheuer H.-P., Weisshart K., Favre A., Lavrik O.I. (2000) Synthesis of base-substituted dUTP analogues carrying a photoreactive group and their application to study human replication protein A. Bioconjugate Chemistry, 11, 445-451.
• Kolpashchikov D.M., Alexandrova L.A., Zakirova N.F., Khodyreva S.N., Lavrik O.I. (2000) Synthesis of photoreactive analog of 2',3'-dideoxyuridine-5'-triphosphate and its usage for photoaffinity modification of human replication factor A. Russian Journal of Bioorganic Chemistry, 26, 134-137.
• Rechkunova N.I., Kolpashchikov D.M., Lebedeva N.A., Petruseva I.O., Dobrikov M.I., Degtyarev S.K., Lavrik O.I. (2000) Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents. Biochemistry (Moscow), 65,244-249.
•  Kolpashchikov D.M., Pestryakov P.E., Wlassoff W.A., Khodyreva S.N., Lavrik O. (2000) Study of interaction of human replication factor A with DNA using new photoreactive analogs of dTTP. Biochemistry (Moscow), 65, 160-163.
• Lavrik O.I., Kolpashchikov D.M., Weisshart K., Nasheuer H.-P., Khodyreva S.N., Favre A. (1999) RPA subunit arrangement near the 3´-end of the primer is modulated by the length of the template-strand and cooperative protein interactions. Nucleic Acids Research, 27, 4235-4240.
• Kolpashchikov D.M., Zakharenko A.L., Dezhurov S.V., Rechkunova N.I., Khodyreva S.N., Degtyarev S.Kh., Litvak V.V., Lavrik O.I. (1999) New reagents for directed modification of biopolymers: photoaffinity modification of Tte DNA polymerase. Russian Journal of Bioorganic Chemistry, 25, 110-117.
• Morozova O.V., Kolpashchikov D.M., Ivanova T.M., Godovikova T.S. (1999) Synthesis of new photocross-linking 5-C-base-substituted UTP analogs and their application in highly selective affinity labelling of the tick-borne encephalitis virus RNA replicase proteins. Nucleosides & Nucleotides, 18, 1513-1514.
• Kolpashchikov D.M., Weisshart K., Nasheuer H.-P., Khodyreva S.N., Fanning E., Favre A., Lavrik O.I. (1999) Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA. FEBS Letters, 450, 131-134.
• Godovikova T.S., Kolpashchikov D.M., Orlova T.N., Richter V.A., Ivanova T.M., Grochevsky S.L., Nasedkina T.V., Poletaev A.I. (1999) 5-{3-[N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-amino]-trans-propenyl-1}-2'-deoxyuridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction. Bioconjugate Chemistry, 10, 529-537.
• Kolpashchikov D.M., Rechkunova N.I., Dobrikov M.I., Khodyreva S.N., Lebedeva N.A., Lavrik O.I (1999) Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases. FEBS Letters, 448,141-144.
• Lavrik O.I., Kolpashchikov D.M., Nasheuer H.-P., Weisshart K., Favre A. (1998) Alternative conformations of human replication protein A are detected by crosslinks with primers carrying photoreactive group at 3'–end. FEBS Letters, 441, 186-190.

Patents

• Kolpashchikov D.M. (2006) Binary deoxyribozyme probe for highly selective and highly sensitive nucleic acid analysis. COL001-008PCT, Columbia University.
• Kolpashchikov D.M. (2006) Binary probes for fluorescent analysis of nucleic acids. COL001-016PCT, Columbia University.

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