Department of Chemistry

York University

Toronto ON , M3J1P3 Canada

Tel: (416) 736-2100 ext. 22817

Fax: (416) 736-5936

Current Projects:

I. Kinetic capillary electrophoresis mediated screening of combinatorial libraries for affinity ligands and drug candidates

Capillary electrophoresis (CE) has become an indispensable technique for the analysis of proteins, nucleic acids, and small molecule compounds. CE was also applied to analyses of biomolecular complexes, determination of binding parameters of ligand-target interactions, and high-throughput screening of combinatorial libraries.

Kinetic capillary electrophoresis (KCE) could be defined as CE separation of molecules, which interact during electrophoresis. Simple association and dissociation kinetics clearly describes the laws of processes and unambiguously predicts the migration of species along the capillary.

We recently introduced two CE-based methods for selection of aptamers: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) and non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Understanding kinetics of interaction allows us not only select ligands, but also implement selection in a “smart” way. We showed that both ECEEM and NECEEM methods could be applied to select “ smart aptamers” – ligands with pre-defined binding parameters ( Kd and koff constants, respectively). This is achieved by collecting a set of fractions, each of which contains species with defined constants according to simple distributions:

ECEEM

NECEEM

where [T] is equlibrium concentration of a target (protein), t (DNA) and t (T-DNA) are migration times (at fixed conditions) of DNA library and intact target-DNA complex, respectively. So that, variable times of collection t determine constants of aptamers in collection windows. This makes CE a unique technique to screen combinatorial libraries of biopolymers and small molecules for ligands with desired binding constants. We anticipate that KCE methods will be widely used in comprehensive screenings of biomolecules.

Recently, we demonstrated that smart aptamers can facilitate multi-probe affinity analysis of a protein with an ultra-wide dynamic range of measured concentrations [Link].In the proof-of-principle work, we used three previously selected smart aptamers for MutS protein. Each aptamer had different Kd and detected MutS concentration in the range of its Kd. The analysis based on a mixture of three aptamers had a dynamic range of more than four orders of magnitude. To the best of our knowledge, this is the widest dynamic range ever reported for affinity analyses of proteins. Intriguingly, this analysis introduces the first “real life” application of smart aptamers.

References:

(1) Drabovich, A.P. ; Okhonin, V.; Berezovski, M.; Krylov, S.N. Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations. Journal of the American Chemical Society 2007 , 129 , 7260-7261. [PDF] This work is featured in Chem. & Eng. News 2007 , 85 (25), 14. [Link]

(2) Berezovski, M.; Musheev, M.U.; Drabovich, A.P. ; Jitkova, J.; Krylov, S.N. Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides. Nature Protocols 2006 , 1 , 1359-1369. [PDF]

(3) Drabovich, A.P. ; Berezovski, M.; Okhonin, V.; Krylov, S.N. Selection of smart aptamers by methods of kinetic capillary electrophoresis. Analytical Chemistry 2006 , 78 , 3171-3178. [PDF]

(4) Berezovski, M.; Musheev, M.; Drabovich, A. ; Krylov S.N. Non-SELEX Selection of Aptamers. Journal of the American Chemical Society 2006, 128 , 1410 -1411. [PDF]

(5) Drabovich, A.P. ; Berezovski, M.; Krylova, S.M.; Musheev, M.U.; Krylov, S.N. Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) as Swiss Army Knife for Selection of Aptamers. P/ACE Setter Newsletter - The Worldwide Newsletter for Capillary Electrophoresis, 2006 , 10 (1), 1- 5. [PDF]

(6) Drabovich, A .; Berezovski, M.; Krylov, S.N. Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Journal of the American Chemical Society 2005 , 127 , 11224-11225. [PDF]

(7) Berezovski, M.; Drabovich, A. ; Krylova, S.M.; Musheev, M.; Okhonin, V.; Petrov, A.; Krylov, S.N. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) – a universal tool for selection of aptamers. Journal of the American Chemical Society 2005 , 127 , 3165-3171. [PDF]

II. Protein-DNA interactions as an analytical tool in capillary electrophoresis

Non-covalent protein-DNA interactions could be classified as binding of natural DNA-binding proteins to oligonucleotides or as an interaction of in vitro selected DNA aptamers with their target proteins. A number of proteins involved in DNA replication, DNA repair, and gene expression are capable of binding DNA and RNA with different affinity and sequence specificity. This ability of DNA- and RNA-binding proteins has a yet-to-be realized potential in analytical sciences. In a similar way, DNA aptamers are considered as “artificial antibodies” for the analysis of proteins both in vitro and in cells.

We endeavour to introduce protein-DNA interactions as a highly efficient tool in a number of CE-based analyses of biopolymers. We have already demonstrated applications of DNA-binding proteins in CE analyses: single-stranded DNA binding protein (SSB) for highly efficient gel-free analysis of products of polymerase-chain reaction (PCR) and DNA mismatch-binding protein (MutS) for identification of base pairs in single-nucleotide polymorphisms (SNP). Furthermore, we plan to use in vitro selected DNA aptamers for quantitative affinity analysis of proteins in cell lysate and in single cells.

We foresee that both DNA-binding proteins and DNA aptamers will be used as toolsets for a variety of analyses of biological molecules.

References:

(8) Liu, Z.; Drabovich, A.P. ; Krylov, S.N.; Pawliszyn, J. Dynamic Kinetic Capillary Isoelectric Focusing: A Powerful Tool for Studying Protein-DNA Interactions . Analytical Chemistry 2007 , 79 , 1097-1100. [PDF]

(9) Drabovich, A.P. ; Krylov, S.N. Identification of Base Pairs in Single-Nucleotide Polymorphisms by MutS Protein-Mediated Capillary Electrophoresis. Analytical Chemistry 2006 , 78 , 2035-2038. [PDF]

(10) Drabovich, A. ; Krylov, S.N. Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis. Journal of Chromatography A 2004 , 1051 , 171-175. [PDF]

Last updated: June 24, 2007