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 ΔT(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

Molecular basis for the inhibition of HMGA1 proteins by distamycin A

The molecular mechanism for the displacement of HMGA1 proteins from DNA is integral to disrupting their cellular function, which is linked to many metastatic cancers. Chemical shift and NOESY NMR experiments provide structural evidence for the displacement of an AT hook peptide (DNA binding motif of HMGA1 proteins) by both monomeric and dimeric distamycin. However, the displaced AT hook alters distamycin binding by weakening the distamycin:DNA complex, while slowing monomeric distamycin dissociation when AT hook is in excess. The central role of the AT hook was evaluated by monitoring full-length HMGA1a protein binding using fluorescence anisotropy. HMGA1a was effectively displaced by distamycin, but the cooperative binding exhibited by distamycin was eliminated by displaced HMGA1a. Additionally, these studies indicate that HMGA1a is displaced from the DNA by 1 equiv of distamycin, suggesting the ability to develop therapeutics that take advantage of the positively cooperative nature of HMGA1a binding

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg(1/2), of ∼3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg(1/2), equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg(2+) concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches ∼1 mM Mg(2+). The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies

Recognition of Specific DNA Sequences by Stacked Pyrrole- and Imidazole- Containing Polyamides: An Efficient Screening Method Based on Competitive Dialysis

A competitive dialysis method has been developed to screen compounds for their DNA binding properties, and it is based on directly comparing the binding of polyamide molecules to a series of distinctively varied, short, synthetic deoxyribonucleotides. Relative binding ratios for each polyamideoligonucleotide pairing were calculated from the concentrations of free polyamide and total polyamide in order to quantitatively compare binding to different DNA sequences. This approach works well as a preliminary screen to determine the viability of novel small molecules, prior to investing significant resources in further characterization and development of possible sequence specific DNA targeted therapeutic agents. The trends in binding affinities of the four triamide molecules (f-ImPyIm, distamycin A, f-PyPyPy and f-ImImPy; where Im is imidazole and Py is pyrrole) correlated well with data obtained from surface plasmon resonance (SPR) studies. Results from circular dichroism studies confirmed the minor groove side-by-side stacked binding motif of the triamides, and thermal stability experiments corroborated the improved DNA stability of promising polyamide-DNA complexes. The affinity of distamycin A for its cognate DNA sequence (A3T3) was unambiguously selected over the other DNA sequences tested. Alternatively, as expected from SPR, circular dichroism and thermal melting experiments, f-ImImPy showed very poor affinity for DNA sequences tested, including its cognate DNA, TCGA. Thus, the very good (distamycin A) and very poor (f-ImImPy) DNA binders were effectively screened

Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm

The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide language in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex

Targeting the inverted CCAAT box 2 in the topoisomerase IIα promoter by \u3cstrong\u3eJH-37\u3c/strong\u3e, an imidazole-pyrrole polyamide hairpin: design, synthesis, molecular biology, and biophysical studies

The topoisomerase IIα 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 IIα 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 IIα promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1 sites. The larger ΔTM values for ICB2,3 (8−9 °C) over ICB1,4,5 (4−5 °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 × 107 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 ΔG° 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 IIα 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

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 \u3e -PyPy- \u3e -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