Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DN A-binding properties of HU histone-like bacterial protein, which is known t o have a low sequence specificity. All 4096 hexamers flanked within 8mers b y degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x100 x 20 mm polyacrylamide gel pads of the microchip, Single-stranded immobilized oligonucleotides were converted in some experiments to the doub le-stranded form by hybridization with a specified mixture of 8mers. The DN A interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curv es of complexes of labeled HU with single-stranded microchip oligonucleotid es; (iii) the effect of HU binding on melting curves of microchip double-st randed DNA labeled with another fluorescent dye, Texas Red, Large numbers o f measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluore scence microscope. Statistical analysis of these data suggests some prefere nce for HU binding to G/C-rich single-stranded oligonucleotides. HU complex es with double-stranded microchip 8mers can be divided into two groups in w hich HU binding either increased the melting temperature (T-m) of duplexes or decreased it. The stabilized duplexes showed some preference for presenc e of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for lon ger stretches of A/T duplexes. Binding of HU to labeled duplexes in the sec ond type of complex caused some decrease in fluorescence. This decrease als o correlates with the higher A/T content and lower T-m. The results demonst rate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.