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.

Regulation of Lipid Biosynthesis, Sliding Motility, and Biofilm Formation by a Membrane-Anchored Nucleoid-Associated Protein of Mycobacterium tuberculosis

“…These structures presumably formed due to the cooperative binding of the protein on DNA. Self-association, DNA-dependent oligomerization, and sequence-independent DNA binding are the characteristic features of many NAPs (29,30). In addition to the DNA binding properties, the abundance of the NAPs in the cells contributes to the compact organization of the genome.…”

“…Briefly, the M. tuberculosis H37Ra cells were grown to an optical density at 600 nm (OD 600 ) of 0.6, corresponding to the mid-log phase (day 5) of growth, and cells were suspended in lysis buffer and boiled for 45 min. The total cell lysate was subjected to SDS-PAGE, along with different amounts of purified Rv3852 (40,30,20, and 15 ng), and detected by immunoblotting with anti-Rv3852 antibody. Quantitation of band intensities was carried out by scanning the bands and analyzing the image with Image Gauge V2.54 software.…”

Quantitative prediction of NF-κB DNA– protein interactions

“…However, it is desirable that the number of data points should be large enough to reduce prediction error because of experimental variation. With microarrays, it is possible to assay the training set in a single experiment (12,13). In theory, data could also be acquired from crystallographic or NMR studies generated for a single site (25), but data from many structures would be needed to match the predictive power of the model presented here.…”

“…Finally, it is not always possible to construct a multiple alignment of binding sites, because there may be no consistent choice for the sequences' orientations such that every pair of sequences is optimally aligned. This is a serious problem in microarray (12,13) and SELEX (14,15) studies, where all binding sites are tested. SELEX uses a pool of random sequences to assay binding but tends to provide little information about low-affinity sites, which are important in multifactor complexes.…”

Evaluation of Gel-Pad Oligonucleotide Microarray Technology by Using Artificial Neural Networks

“…This level of discrimination is needed to resolve variants of highly conserved genes (e.g., those encoding the rRNAs). Numerous studies have been conducted using gel-pad microarrays (8,11,12,20,23,24,26,45,47,48,49) because melting profiles of probe-target duplexes are thought to offer better discrimination between target and nontarget sequences than planar microarrays, which typically depend on signal intensity (SI) values obtained following hybridization and wash conditions adjusted to an appropriate (average) stringency. (The term "melting profile," used throughout this article, refers to nonequilibrium dissociation curves.)…”