title: UV fluorescence of tryptophan residues effectively measures protein binding to nucleic acid fragments immobilized in gel elements of microarrays.
abstract: 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.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia. lana@biochip.ru.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Zasedateleva
authors.forename: Vadim A
authors.initials: VA
authors.lastname: Vasiliskov
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Surzhikov
authors.forename: Alexey Y
authors.initials: AY
authors.lastname: Sazykin
authors.forename: Lidiya V
authors.initials: LV
authors.lastname: Putlyaeva
authors.forename: Anton M
authors.initials: AM
authors.lastname: Schwarz
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Kuprash
authors.forename: Alla Y
authors.initials: AY
authors.lastname: Rubina
authors.forename: Victor E
authors.initials: VE
authors.lastname: Barsky
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Zasedatelev
mesh_terms: D005453:Fluorescence; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D061925:Immobilized Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D019169:Jurkat Cells; D046228:Microarray Analysis / Q000379:methods; D050778:NFATC Transcription Factors / Q000378:metabolism*; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D061925:Immobilized Nucleic Acids; D050778:NFATC Transcription Factors; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D014364:Tryptophan
keywords: Gel-based microarrays; Protein-nucleic acid interactions; UV fluorescence microscope; UV fluorescence of proteins