Shape readout of AT‐rich DNA by carbohydrates

Abstract

Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove-binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA “shape readout” properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT-rich) DNA. Isothermal Titration Calorimetry (ITC), Circular Dichroism (CD) titration, Ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT-rich DNA duplex d[5′-G2A6T6C2-3′]. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two-binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy driven with a binding constant of approximately 108 M−1. ITC-derived binding enthalpies were used to obtain the binding-induced change in heat capacity (ΔCp) of -225±19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT-tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT-rich DNA (d[5′-G2A6T6C2-3′]) > B form alternate AT-rich DNA (d[5′-G2(AT)6C2-3′]) > A form GC-rich DNA (d[5′-A2G6C6T2-3′]), demonstrating the preference of ligand 1 for B* form DNA.

Rapid Synthesis, RNA Binding, and Antibacterial Screening of a Peptidic-Aminosugar (PA) Library

“…The assay was validated for A-site binding by determining the relative displacement of known aminoglycosides with previously reported results; 40 additionally, the assay has been successfully employed in screening of a neomycin dimer compound library. 18 …”

“…The results of the assay were well correlated with antibacterial activity of bacterial strains determined by MIC calculations. 18 …”

Influence of linker length in shape recognition of B∗ DNA by dimeric aminoglycosides

“…Hexa- N -Boc deoxy-neomycin-5″-amine with short and long (Figure 1) linkers were synthesized from commercially available neomycin sulphate using previously published methods. 13, 18, 19, 21, 22 The diisothiocyanate linkers used in the synthesis of neomycin dimers were prepared by reacting commercially available diamines with TCDP (1,1′-thiocarbonyldi-2(1 H )-pyridone) in almost quantitative yields (see supplementary information Table S1). The Hexa- N -Boc deoxy-neomycin-5″-amines were then reacted with diisothiocyanates and diisocyanates in the presence of pyridine (dry) and DMAP (catalytic amounts) for 12 hours at room temperature to produce the Boc protected neomycin dimers with various linker lengths (DPA71–DPA80, see supporting information Scheme S1).…”

“…The ITC studies were performed at pH = 5.5 to ensure that the dimers were substantially protonated (lowest pK a for an amine group of neomycin = 5.91). 19 The following observations were noted from the thermodynamic characterization. (1) Similar to our earlier reported work, neomycin dimers with linker length L=12 to 22 exhibit two sets of binding sites, in which the first is predominantly entropically driven, with a binding stoichiometry of 1:1 (neomycin dimer:DNA) while the second is enthalpically driven with a binding stoichiometry of ~3:1 (neomycin dimer:DNA).…”

An overview of recent advances in duplex DNA recognition by small molecules

“…18 ) binds to short oligonucleotides (12 mer) with 1:1 ligand to DNA duplex stoichiometry and show a binding site size of 11–12 base pairs with the polymeric DNA. A complete thermodynamic study of neomycin dimer 115 binding to a B-DNA sequence revealed that the first binding event (the high affinity site) is entropically driven and that the ionic strength dependence of the binding is highly dependent on the electrolytic contribution [ 194 ]. The neomycin dimers also displayed length dependent shape recognition of the B-DNA [ 195 ].…”