Molecular recognition of DNA base pairs by the formamido/pyrrole and formamido/imidazole pairings in stacked polyamides.

Polyamides containing an N-terminal formamido (f) group bind to the minor groove of DNA as staggered, antiparallel dimers in a sequence-specific manner. The formamido group increases the affinity and binding site size, and it promotes the molecules to stack in a staggered fashion thereby pairing itself with either a pyrrole (Py) or an imidazole (Im). There has not been a systematic study on the DNA recognition properties of the f/Py and f/Im terminal pairings. These pairings were analyzed here in the context of f-ImPyPy, f-ImPyIm, f-PyPyPy and f-PyPyIm, which contain the central pairing modes, -ImPy- and -PyPy-. The specificity of these triamides towards symmetrical recognition sites allowed for the f/Py and f/Im terminal pairings to be directly compared by SPR, CD and DeltaT (M) experiments. The f/Py pairing, when placed next to the -ImPy- or -PyPy- central pairings, prefers A/T and T/A base pairs to G/C base pairs, suggesting that f/Py has similar DNA recognition specificity to Py/Py. With -ImPy- central pairings, f/Im prefers C/G base pairs (>10 times) to the other Watson-Crick base pairs; therefore, f/Im behaves like the Py/Im pair. However, the f/Im pairing is not selective for the C/G base pair when placed next to the -PyPy- central pairings.

Extending the language of DNA molecular recognition by polyamides: unexpected influence of imidazole and pyrrole arrangement on binding affinity and specificity.

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity has been investigated using DNA melting (DeltaT(M)), circular dichroism (CD), and surface plasmon resonance (SPR) studies. SPR results obtained from a complete set of triheterocyclic polyamides show a dramatic difference in the affinity of f-ImPyIm for its cognate DNA (K(eq) = 1.9 x 10(8) M(-1)) and f-PyPyIm for its cognate DNA (K(eq) = 5.9 x 10(5) M(-1)), which could not have been anticipated prior to characterization of these compounds. Moreover, f-ImPyIm has a 10-fold greater affinity for CGCG than distamycin A has for its cognate, AATT. To understand this difference, the triamide dimers are divided into two structural groupings: central and terminal pairings. The four possible central pairings show decreasing selectivity and affinity for their respective cognate sequences: -ImPy > -PyPy- >> -PyIm- approximately -ImIm-. These results extend the language of current design motifs for polyamide sequence recognition to include the use of "words" for recognizing two adjacent base pairs, rather than "letters" for binding to single base pairs. Thus, polyamides designed to target Watson-Crick base pairs should utilize the strength of -ImPy- and -PyPy- central pairings. The f/Im and f/Py terminal groups yielded no advantage for their respective C/G or T/A base pairs. The exception is with the -ImPy- central pairing, for which f/Im has a 10-fold greater affinity for C/G than f/Py has for T/A.

Targeting the inverted CCAAT box 2 in the topoisomerase IIalpha promoter by JH-37, an imidazole-pyrrole polyamide hairpin: design, synthesis, molecular biology, and biophysical studies.

The topoisomerase IIalpha promoter is regulated through transcription factor interactions with five inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 represses the expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites within the topoisomerase IIalpha promoter are, therefore, potential targets for the design of anticancer drugs and gene control agents. The synthesis and DNA binding properties of a hairpin polyamide molecule (JH-37) that targets 5'-TTGGT-3' found in ICB2 and ICB3 sites are described. Gel shift and DNase I footprinting studies on the topoisomerase IIalpha promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1 sites. The larger DeltaT(M) values for ICB2,3 (8-9 degrees C) over ICB1,4,5 (4-5 degrees C) indicated a preference of JH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1 binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 x 10(7) M(-1)), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). The improved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferred site. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpin polyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the DeltaG degrees of binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamic and kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised to systematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivity for ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, but multiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sites in the topoisomerase IIalpha promoter is an ideal test for this strategy. This approach is in contrast to the traditional strategy of targeting 15-16 base pairs, which has not been successful in actual biological systems due to poor cell uptake and distribution.