Sequence specificity of dimeric bisbenzimidazoles to AT-sequences of DNA of different nucleotide composition determined by footprinting

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Abstract

The study aimed to investigate the site-specificity of binding to DNA of three series of minor groove ligands – dimeric bisbenzimidazoles DB2(n), DB2P(n), and DB2Py(n) – using DNAase I footprinting. The compounds consist of two bisbenzimidazole units linked by oligomethylene linkers of varying lengths (n), with structural modifications to enhance DNA-binding properties. The binding specificity of the compounds was determined using DNAase I footprinting. The DB2(n) and DB2P(n) series are analogs of Hoechst 33342, modified by removing hydrophobic ethoxyphenol cores and introducing hydrophilic aminomethylene groups. The DB2Py(n) series incorporates a pyrrolcarboxamide group, a structural unit of the AT-specific antibiotic netropsin. The interaction of these compounds with DNA sequences was analyzed to identify their binding preferences. All studied compounds demonstrated specificity for AT-rich DNA sequences. The DB2P(n) and DB2(n) series exhibited increased affinity for (AATT)3 and TTTT sequences. The DB2Py(n) series showed high specificity to AT-rich regions, with a preference for the TTTT motif. None of the compounds interacted with sequences containing fewer than four AT base pairs. These findings highlight the influence of structural modifications on DNA-binding specificity and affinity. The study revealed that dimeric bisbenzimidazoles DB2(n), DB2P(n), and DB2Py(n) exhibit distinct binding preferences for AT-rich DNA sequences, with DB2Py(n) showing a pronounced affinity for the TTTT motif. The results demonstrate the potential of these compounds as tools for targeting specific DNA sequences, with implications for molecular biology and drug design.

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About the authors

D. S. Naberezhnov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991; Kashirskoe shosse 24, Moscow, 115522

A. F. Arutuynyan

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

A. D. Beniaminov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

N. M. Smirnov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

D. N. Kaluzhny

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

A. L. Zhuze

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991

O. Y. Susova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health

Author for correspondence.
Email: susovaolga@gmail.com
Russian Federation, ul. Vavilova 32, Moscow, 119991; Kashirskoe shosse 24, Moscow, 115522

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Supplementary files

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2. Fig. 1. Structural formulas of the studied narrow groove ligands.

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3. Fig. 2. Profiles of DNase I cleavage of fluorescently labeled d150T PCR fragments in the presence of monomeric and dimeric bisbenzimidazoles. 0 – initial 150-bp PCR fragment; K – DNase I cleavage in the absence of narrow groove ligands; T – chemical cleavage at thymines; HT – cleavage in the presence of HT, MB2 – cleavage in the presence of MB2, DB2(6) – cleavage in the presence of DB2(6), DB2P(1) – cleavage in the presence of DB2P(1).

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4. Fig. 3. Profiles of DNase I cleavage of fluorescently labeled d150T PCR fragments in the presence of DB2P(n) series compounds. 1 (K) – initial 150-bp PCR fragment; 2 (T) – chemical cleavage at thymines; 3 (0) – DNase I cleavage in the absence of narrow groove ligands; 4–10 – cleavage in the presence of DB2P(1) taken at concentrations of 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 μM, respectively; 11 – cleavage in the presence of 5 μM DB2P(2); 12 – cleavage in the presence of 5 μM DB2P(3); 13 – cleavage in the presence of 5 μM DB2P(4).

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5. Fig. 4. Profiles of DNase I cleavage of fluorescently labeled d150T PCR fragments in the presence of DB2(n) series compounds.1 (K) – initial 150-bp PCR fragment; 2 (T) – chemical cleavage at thymines; 3 (0) – DNase I cleavage in the absence of narrow groove ligands; 4–10 – cleavage in the presence of DB2(6), taken at concentrations of 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 μM, respectively; 11 – cleavage in the presence of 5 μM DB2(9); 12 – cleavage in the presence of 5 μM DB2(10); 13 – cleavage in the presence of 5 μM DB2(11).

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6. Fig. 5. Profiles of DNase I cleavage of TAMRA-labeled d84AT PCR fragments in the presence of DB2P(1). 1 – original 84-nt PCR fragment; 2 – chemical cleavage at thymines; 3 – DNase I cleavage in the absence of DB2P(1); 4–10 – cleavage in the presence of DB2P(1) taken at concentrations of 0.3, 0.5, 1, 2, 4, 6, and 8 μM, respectively.

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7. Fig. 6. Profiles of DNase I cleavage of FAM-labeled d87KNS PCR fragments in the presence of DB2P(1). 1 – initial 84-nucleotide PCR fragment; 2, 3 – chemical cleavage at adenines and guanines in the presence of formic acid; 4 – DNase I cleavage in the absence of narrow groove ligands; 5–9 – cleavage in the presence of DB2P(1) taken at concentrations of 0.06, 0.3, 1.6, 8, and 40 μM, respectively.

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8. Fig. 7. Selectivity of compounds DB2Py(4) and DB2Py(5) to the nucleotide sequence.

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