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

Abstract

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T(m)) of duplexes or decreased it. The stabilized duplexes showed some preference for presence 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 longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T(m). The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

Figure 1

Non-equilibrium melting curves for complexes of FITC-labeled HU protein with five microchip-immobilized octamers containing specific 6mer cores.

Figure 2

Mean melting temperatures (A) and labeled HU protein binding (B) for oligonucleotides with different numbers of A/T bases in the hexamer core. HU binding is estimated by measuring the fluorescence of labeled HU located in each gel pad of the generic microchip. The standard values and deviations are indicated by columns and bars, respectively.

Figure 3

Non-equilibrium melting curves for a microchip duplex measured in the absence (1) and presence (2) of HU protein. A duplex was formed by hybridization of the microchip oligonucleotide gel–5′-MAGTCTGM-3′ with the fluorescently labeled oligonucleotides 3′-MTCAGACM-5′-TR from the hybridization mixture.

Figure 4

Distribution of the number of oligonucleotides (N) characterized by different ΔT_m. ΔT_m = T_m(+HU) – T_m(–HU).

Figure 5

Average shifts of T_m (ΔT_m) for the 10 best motifs with 3 base long (A) and 4 base long (B) motifs. The standard mean values and deviations of ΔT_m are indicated.

Figure 6

The effect of HU protein binding on fluorescence intensity of labeled microchip dsDNA with different GC contents.

Figure 7

The effect of HU protein on fluorescence of the microchip dsDNA with different ΔT_m. R = fluorescence(+HU)/fluorescence(–HU).