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

Olga A. Zasedateleva, Vadim A. Vasiliskov, Sergey A. Surzhikov, Alexey Y. Sazykin, Lidiya V. Putlyaeva, Anton M. Schwarz, Dmitry V. Kuprash, Alla Y. Rubina, Victor E. Barsky, Alexander S. Zasedatelev

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1074-1080
Number of pages7
JournalBiotechnology Journal
Volume9
Issue number8
DOIs
Publication statusPublished - Aug 2014
Externally publishedYes

Keywords

  • Gel-based microarrays
  • Protein-nucleic acid interactions
  • UV fluorescence microscope
  • UV fluorescence of proteins

Fingerprint

Dive into the research topics of 'UV fluorescence of tryptophan residues effectively measures protein binding to nucleic acid fragments immobilized in gel elements of microarrays'. Together they form a unique fingerprint.

Cite this