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Engineered modular heterocyclic-diamidines for sequence-specific recognition of mixed AT/GC base pairs at the DNA minor groove.

ABSTRACT: This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA. A key goal is to invent synthetic chemistry for compound preparation to recognize an adjacent GG sequence that has been difficult to target. After trying several unsuccessful compound designs, an N-alkyl-benzodiimidazole structure was selected to provide two H-bond acceptors for the adjacent GG-NH groups. Flanking thiophenes provide a preorganized structure with strong affinity, DB2831, and the structure is terminated by phenyl-amidines. The binding experimental results for DB2831 with a target AAAGGTTT sequence were successful and include a high ΔT _m, biosensor SPR with a K _D of 4 nM, a similar K _D from fluorescence titrations and supporting competition mass spectrometry. MD analysis of DB2831 bound to an AAAGGTTT site reveals that the two unprotonated N of the benzodiimidazole group form strong H-bonds (based on distance) with the two central G-NH while the central -CH of the benzodiimidazole is close to the -C[double bond, length as m-dash]O of a C base. These three interactions account for the strong preference of DB2831 for a -GG- sequence. Surprisingly, a complex with one dynamic, interfacial water is favored with 75% occupancy.

SUBMITTER: Guo P

PROVIDER: S-EPMC8672716 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The classical model of DNA minor groove binding compounds is that they should have a crescent shape that closely fits the helical twist of the groove. Several compounds with relatively linear shape and large dihedral twist, however, have been found recently to bind strongly to the minor groove. These observations raise the question of how far the curvature requirement could be relaxed. As an initial step in experimental analysis of this question, a linear triphenyl diamidine, DB1111, and a series of nitrogen tricyclic analogues were prepared. The goal with the heterocycles is to design GC binding selectivity into heterocyclic compounds that can get into cells and exert biological effects. The compounds have a zero radius of curvature from amidine carbon to amidine carbon but a significant dihedral twist across the tricyclic and amidine-ring junctions. They would not be expected to bind well to the DNA minor groove by shape-matching criteria. Detailed DNase I footprinting studies of the sequence specificity of this set of diamidines indicated that a pyrimidine heterocyclic derivative, DB1242, binds specifically to a GC-rich sequence, -GCTCG-. It binds to the GC sequence more strongly than to the usual AT recognition sequences for curved minor groove agents. Other similar derivatives did not exhibit the GC specificity. Biosensor-surface plasmon resonance and isothermal titration calorimetry experiments indicate that DB1242 binds to the GC sequence as a highly cooperative stacked dimer. Circular dichroism results indicate that the compound binds in the minor groove. Molecular modeling studies support a minor groove complex and provide an inter-compound and compound-DNA hydrogen-bonding rational for the unusual GC binding specificity and the requirement for a pyrimidine heterocycle. This compound represents a new direction in the development of DNA sequence-specific agents, and it is the first non-polyamide, synthetic compound to specifically recognize a DNA sequence with a majority of GC base pairs.

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Engineered modular heterocyclic-diamidines for sequence-specific recognition of mixed AT/GC base pairs at the DNA minor groove.

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