Quantitative Microarray Profiling of DNA-Binding Molecules

Abstract

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5′-TACGT-3′within the hypoxia response element (HRE) yielded a CSI microarrayderived sequence motif of 5′-WWACGT-3′ (W = A,T). A linear β-linked polyamide programmed to target a (GAA) 3 repeat yielded a CSI microarray-derived sequence motif of 5′-AARAARWWG-3′ (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of K a values across the microarray intensity spectrum.

Specificity landscapes of DNA binding molecules elucidate biological function

“…3A and Fig. S3) (30). The SSL of this bioactive molecule validates the increased preference for Thymine by the thiophene ring.…”

“…Examination of the outer-ring peaks identified three related submotifs (Fig. 3B) that were concealed in consensus motifs derived from PWMs (30). This example of relaxed specificity displayed by a linear polyamide (PA-5), along with the proteins described above, serve to highlight the ability of SSLs to identify multiple binding submotifs that are compressed into a single consensus by motiffinding algorithms.…”

“…SSLs directly translate to binding energy landscapes for hairpin polyamides because their CSI intensities correlate strongly with equilibrium binding energies measured in solution (29,30). To evaluate if this relationship holds for a natural DNA binding protein, we measured affinities of Nkx-2.5 for six DNA sequences that span a range of CSI intensities.…”

Development of a molecule-recognized promoter DNA sequence for inhibition of HER2 expression

“…Alternatively, improvement of specificity by a combinatorial modification of the PI polyamide-HER2 compound may be needed. 27,28 These observations indicate that PI polyamide-HER2 induces the decrease of HER2 mRNA expression and consequently inhibits cell proliferation in various cancer cells, including MDA-MB-231 cells. As far as we know, this is the first report that a chemical reagent decreases HER2 transcription in cancer cell and inhibits cell growth in various cancer cell lines.…”

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

“…[25][26][27][28][29] Sequence specificities of Py-Im polyamides have also been investigated by using high-throughput sequencing. [30][31][32] Here, we designed and synthesized thirteen Py-Im polyamides ( Fig.…”