537049-66-4Relevant articles and documents
Redirection of the Transcription Factor SP1 to AT Rich Binding Sites by a Synthetic Adaptor Molecule
Bolz, Mathias,Scheffer, Ute,Kalden, Elisabeth,G?bel, Michael W.
, (2021/08/25)
The ubiquitous transcription factor SP1 binds to a GC rich consensus sequence. Here we describe an adaptor molecule that mediates binding of SP1 to a non-cognate DNA site rich in AT. The adaptor is comprised of a Dervan-type hairpin polyamide with high affinity to an AT rich hexamer duplex. It also carries a 27mer DNA that contains the SP1 consensus sequence. The synthesis and purification of the polyamide-DNA conjugate is reported. Pulldown experiments and western blot analysis demonstrate adaptor mediated binding of SP1 to the hexamer duplex TTGTTA.
Sequence specific and high affinity recognition of 5′-ACGCGT-3′ by rationally designed pyrrole-imidazole H-pin polyamides: Thermodynamic and structural studies
Mackay, Hilary,Brown, Toni,Uthe, Peter B.,Westrate, Laura,Sielaff, Alan,Jones, Justin,Lajiness, James P.,Kluza, Jerome,O'Hare, Caroline,Nguyen, Binh,Davis, Zach,Bruce, Chrystal,David Wilson,Hartley, John A.,Lee, Moses
experimental part, p. 9145 - 9153 (2009/04/11)
Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f = formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5′-ACGCGT-3′ through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6 × 108 M-1 and 2.0 × 1010 M-1 for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (ΔH of -3.3 and -1.0 kcal mol-1 for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (ΔCp) were determined to be -116 and -499 cal mol-1 K-1, respectively. These results are in general agreement with ΔCp values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5′-CCACGCGTGG)2. According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.
