Dmitry Kolpashchikov

Assistant Professor of Chemistry Phone:407-823-6752 Fax: 407-823-2252 E-mail: dkolpash@mail.ucf.edu Website: http://columbiamedicine2.org/CPET/people/dmitry.html

Research

Our research interests cover all aspects of chemistry and biochemistry of nucleic acid and nucleic acid binding proteins.In particular, two following projects are in progress: Development of probes for nucleic acid analysis and Nucleic acid - based logic circuits.

Probes for nucleic acid analysis

Numerous techniques for DNA/RNA analysis rely on the ability of the probe to recognize nucleic acid sequences specifically by forming duplexes. The formation of at least 15-20 nucleotide hybrids between the probe and the analyte is required to uniquely define a specific fragment in a nucleic acid the size of a genome. Hybrids of such length are too stable to be sensitive to a base miss-pairing; since a single mismatch unit results in a relatively small energetic penalty. We have demonstrated that two-component probes can improve selectivity of nucleic acid recognition. Each subunit of such probes binds to a relatively short (7-10 nucleotides) of analyte. These short hybrids are extremely sensitive to a single base substitution even at mild conditions.

One example of binary probe for nucleic acid analysis is binary DNA probe (Figure 2). 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 1, left) and the fluorescent signal is low. Addition of A20 DNA analyte triggers the formation of a quaternary complex (Figure 1, right). The fluorophore (FAM) is remote from the quencher (Dabcyl) in this complex; this 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.

Figure 1. Binary DNA probe. Two oligonucleotide and a molecular beacon exist in a dissociated state in the absence of a DNA analyte (left). Addition of a target DNA analyte results in the formation of fluorescent four-way DNA junction-like structure (right).

Nucleic acid - based logic circuits

Miniaturization of silicon computers will inevitably bring the insurmountable problems of logic element cross-talking and heat dissipation. It is believed that bottom-up organization of molecular circuits is a promising alternative to the modern fabrication of semiconductor microprocessors by photolithography. This bottom-up approach has a potential of bringing to the market a molecular computer, which is smaller and consumes less energy than its electronic counterpart. Our long-term goal is to construct a molecular scale processor basing on oligodeoxyribonucleotide logic gates. One of the biggest shortcomings of existing molecular scale logic gates is the lack of universal large scale connectivity. In addition, localization of functional molecules in chosen nano-environments remains a challenging task. We are trying to solve these two problems.

On the one hand BDP (Figure 1) is a probe for recognition of specific DNA/RNA sequences; on the other hand, it functions as YES logic unit. It should be noted that the YES unit recognized an oligonucleotide input (analyte) and generated an oligonucleotide fragment made of two molecular beacon-binding arms as an output signal. The output is readout by the MB, but can also be recognized by the next gate as an input. We design a basic set of connectable DNA logic gates (NOT, AND, and OR)? Organize them in a network which corresponds to EX-OR logic function both in solution and on a two-dimensional (2D) DNA platform.

Selected Publications

1. Kolpashchikov D.M. (2008). "Split DNA enzyme for visual single nucleotide polymorphism typing."JACS, 130, 2934-2935.
2. Kolpashchikov D.M. (2007) "A binary deoxyribozyme for nucleic acid analysis."ChemBioChem 8, 2039-2042.
3. Kolpashchikov D.M. (2006) "A binary DNA probe for highly specific nucleic acid recognition."JACS, 128, 10625-10628.
4. Kolpashchikov D.M. (2005) "Binary malachite green aptamer for fluorescent detection of nucleic acids. JACS, 127, 12442-12443.
5. Kolpashchikov D.M., Stojanovic M.N. (2005) "Boolean control of aptameric binding states."JACS, 127, 11348-11341.
6. 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.
7. Kolpashchikov D.M. (2003) "Superaffinity labeling of proteins: approaches and techniques."J. Biomol. Struct. Dyn., 21, 55-64.

Federal Grants

1. NGRI R21 "Deoxyribozyme technology for nucleic acid analysis"2006-2009.

Students

Motivated, open-minded students are welcome in our lab. Students will be involved in all aspects of scientific research including literature search, experiment design, data collection and analysis, poster and oral presentations, publishing in scientific journals. Techniques that you will be exposed in our lab include gel electrophoresis, UV/vis and fluorescent spectroscopy, SELEX, bioorganic synthesis (including DNA synthesis), chromatography (including HPLC), photochemistry and others.