Vadim A. Vasiliskov

Kinetics of hybridization on the oligonucleotide microchips with gel pads.

Abstract The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.

UV fluorescence of tryptophan residues effectively measures protein binding to nucleic acid fragments immobilized in gel elements of microarrays.

Microarrays allow for the simultaneous monitoring of protein interactions with different nucleic acid (NA) sequences immobilized in microarray elements. Either fluorescently labeled proteins or specific fluorescently labeled antibodies are used to study protein–NA complexes. We suggest that protein–NA interactions on microarrays can be analyzed by ultraviolet (UV) fluorescence of tryptophan residues in the studied proteins, and this approach may eliminate the protein‐labeling step. A specialized UV microscope was developed to obtain fluorescent images of microarrays in the UV wavelengths and to measure the fluorescence intensity of individual microarray elements. UV fluorescence intensity of BSA immobilized in microarray gel elements increased linearly with increased BSA amount with sensitivity of 0.6 ng. Real‐time interaction curves between the DNA‐binding domain of the NFATc1 transcription factor (NFATc1‐DBD) and synthetic hairpin‐forming oligodeoxyribonucleotides immobilized within 0.2 nL microarray gel elements at a concentration 5 × 10–5 M and higher were obtained. The UV fluorescence intensities of microarray gel elements containing NFATc1‐DBD–DNA complexes at equilibrium allowed the estimation of the equilibrium binding constant for complex formation. The developed method allows the protein–NA binding to be monitored in real time and can be applied to assess the sequence‐specific affinity of NA‐binding proteins in parallel studies involving many NA sequences.

Separate production of single-stranded DNA is not necessary: circuit denaturation of double-stranded DNA followed by hybridization of single strands on oligonucleotide microchips.

Abstract An approach to circuit renaturation-hybridization of dsDNA on oligonucleotide microchips is described. A close circuit cycling device has been developed, and the feasibility of the proposed technique was demonstrated on two platforms. First, a commercial microchip for detection of rifampicin resistance in Mycobacterium tuberculosis was used. Hybridization of a 126 nt long single-stranded DNA (ssDNA) fragment of the rpoB gene according to manufacturers protocol has been compared to hybridization of the same double-stranded DNA (dsDNA) fragment using the developed approach. Hybridization signals obtained by both methods were comparable in intensity and correlated closely. Second, a 22 nt long hairpin-forming oligonucleotide was designed and hybridized with a custom microchip containing probes complementary to both strands of the oligonucleotide. Conventional hybridization of this oligonucleotide did not yield any significant signals. Cleavage of the hairpin loop resulted in the formation of a 9 bp long intermolecular duplex. Hybridization of the duplex using the suggested technique yielded strong signals. The proposed approach allows analyzing target DNA in double-stranded form bypassing the preparation of single-stranded targets. Moreover, both complementary chains could be analyzed simultaneously, providing a reliable internal control. Being combined with fragmentation this method opens new possibilities in analyzing ssDNA with complex secondary structure.

dUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase

Abstract To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3− and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3′ ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3′-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3′-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3′ terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3′–5′ exonuclease functions to cleave the 3′ terminal thymidine in the absence of pyrophosphate.

Bridge DNA amplification of cancer-associated genes on cross-linked agarose microbeads

AbstractA cross-linked agarose substrate was studied as a 3D support for bridge solid-phase DNA amplification (SPA). In this kind of SPA, primers are immobilized on agarose beads. Flow cell studies of SPA in real-time experiments showed that the amplification efficiency is strongly affected by (a) the presence of a linker between the primer and substrate, and (b) by the loading with primers. In fact, a high loading density may compromise SPA. The analysis of real time SPA curves using geometric growth model highlighted the advantage of 3D agarose support over the flat surfaces. The potential of bridge 3D SPA in DNA diagnostics was successfully demonstrated by on-chip analysis of mutations of the cancer-associated genes BRCA1/2 and CHEK2. Graphical AbstractA cross-linked agarose substrate was studied as a 3D support for bridge solid-phase DNA amplification (SPA). In this kind of SPA, primers are immobilized on agarose beads. The potential of bridge 3D SPA in DNA diagnostics was successfully demonstrated by on-chip analysis of mutations of the cancer-associated genes BRCA1/2 and CHEK2.

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.