Sequence specific and high affinity recognition of 5′-ACGCGT-3′ by rationally designed pyrrole-imidazole H-pin polyamides: Thermodynamic and structural studies

Abstract

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.

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole‐Imidazole Polyamides

“… To promote the antiparallel alignment of two PIP strands, PIPs that have two strands joined by linkers have been widely used. Structures such as cyclic‐, [10] hairpin‐, [11,12] and H‐pin‐type PIPs have been proposed to date (Figure 1 and Figure 2 a) [13,14] . Hairpin‐type PIPs are the primary choice because of their ease of synthesis by solid‐phase synthesis, [15,16] but cyclic‐type PIPs were reported to have better binding affinity and selectivity than hairpin‐type PIPs [17–20] …”

“…a) [13,14] . Hairpin‐type PIPs are the primary choice because of their ease of synthesis by solid‐phase synthesis, [15,16] but cyclic‐type PIPs were reported to have better binding affinity and selectivity than hairpin‐type PIPs [17–20] …”

“…a) Chemical structure of hairpin and H-pin PIPs. [12,14] Two PIP strands are joined by a linker moiety promoting an antiparallel alignment in the minor groove of duplex DNA. b) Base-pair recognition by PIPs.…”

Selective binding of small molecules to DNA: Application and perspectives

“…DNA binder can be categorized into compounds that bind noncovalently and those that bind covalently to DNA, and it is interesting that within both categories some compounds can further bind to DNA sequences selectively [5][6][7].…”

Facile Dimer Synthesis for DNA-Binding Polyamide Ligands

“…Due to the increasing complexity and length of recently reported DNA-binding ligands, faster methods to access these increasingly complicated molecules in fewer steps would save both time and purification effort. Furthermore, the continued use of solution-based synthesis for these ligands in recent years − demonstrates that significant room remains for improving their synthetic accessibility with use of SPPS.…”