Book of Abstracts: Albany 2007
June 19-23 2007
New Highly Fluorescing bis-Hoechsts: Physicochemical, Biochemical and Cytological Studies
The goal of our work was design of site-specific ligands covering one turn of double-stranded DNA and recognizing two sites composed of three or four A·T pairs separated by two base pairs including G·C pairs (-(A/T)n-NN-(A/T)n-, where n - 3 or 4). Using computer modeling three new families of covalent dimers of the AT-specific fluorescent dye Hoechst 33258, namely, bis-HT(I), bis-HT(II) and DBBI were constructed and then synthesized (1, 2, 3).
The length of linker R joining two Hoechst molecules was varied within the bis-HT (I) family. We replaced the central CH2 group of bis-HT(I)-7 by a positive N+H(CH3) group with the hope to improve bis-HT(I) solubility. With the same aim, we synthesized the compounds of bis-HT(II) family bearing at the ends N,N-dimethylpropylcarboxamide groups instead of N-methylpiperazine ones. In addition, CH2NHC(O) groups were introduced in the linker R of DBBI family in place of PhO groups that composed linkers R in bis-HT(I) and bis-HT(II) molecules.
Bis-HT(I)-5, -7, and -8 were used for differential chromosome staining from human promonocytic leukemia HL-60 cells (2). The contrast range was the highest for bis-HT (I)-7 and looked similar in quality to the pattern observed for chromosome staining with DAPI (4',6-diamidino-2-phenylindole) dye. It was found that the fluorescence brightness of live and fixed cells stained with bis-HT(I)-5, or -7 only slightly differed, whereas nuclei of live cells stained with bis-HT(I)-8 shone considerably weaker than those of fixed cells. Hence, bis-HT(I)-5, -7, and -8 are new promising fluorescent dyes capable of both differential staining of chromosomes and penetrating through cell and nuclear membranes followed by effective staining of cell nuclei.
The effect of bis-HT(I) and bis-HT(II) on catalytic activity of HIV-1 integrase, one of the three key enzymes required for virus replication, was studied. This enzyme is responsible for incorporation of viral DNA into the host genome and is an attractive target for HIV-1 therapy. It was found that compounds bis-HT (I) and bis-HT(II) inhibit 3?-processing catalyzed by HIV-1 integrase at micro- and submicromolecular concentrations (3). A 21-member oligonucleotide duplex mimicking a terminal region of the U5 LTR fragment of HIV-1 DNA was used as integrase substrate in this reaction. The bis-HT(II)-5, -7, and -8 molecules were nearly one order of magnitude more active than the corresponding homologous bis-HT(I)-5, -7, -8. It is noteworthy that Hoechst 33258 did not inhibit this reaction up to the concentration of 100 μM.
Interaction of one of the synthesized bis-HT(I)-7(NMe) with dsDNA was studied using fluorescent and UV-VIS spectroscopy, circular and linear dichroism (4). It was shown that bis-HT(I)-7(NMe) forms complexes of three types. The first type complexes correspond to binding of bis-HT(I)-7(NMe) monomer in the open conformation; in this case the ligand covers the total dsDNA turn and is located in the minor grove. These complexes are characterized by positive CD at 362 nm and more than 100-fold increase in the fluorescence intensity at 475 nm. It corresponds to a specific type complex of Hoechst 33258 with poly[d(A-T)]·poly[d(A-T]. An ability of bis-HT(I)-7(NMe) in the form of an open monomer to form bridges between two dsDNA molecules, i.e., binding of each of the two bis-HT(I)-7(NMe) ends with two different dsDNA molecule, was demonstrated. This property distinguishes bis-HT(I)-7(NMe) from Hoechst 33258 and may induce a new type of biological activity for the former compound. Indeed, this compound inhibits HIV-1 DNA integrase at the concentrations at least 100 times lower than Hoechst 33258 does. The second type complexes correspond to the bis-HT(I)-7(NMe) dimer binding to dsDNA with stoichiometry ≥ 5 bp (as an inter- or intramolecular sandwich). This type is characterized by maximal positive CD at 345 nm, dramatic decrease in fluorescence intensity, maximum shift to 490 nm, and negative linear dichroism at 360 nm. The binding of this type is associated with the formation of nonspecific dimeric Hoechst 33258-dsDNA complex. The localization of bis-HT(I)-7(NMe) sandwich on dsDNA needs further investigation. The third type complexes correspond to the aggregation Hoechst 33258 complex with dsDNA (stoichiometry approximately 2 bp per bis-HT(I)-7(NMe) molecule). We assume that in these complexes sandwich dimers are formed on dsDNA. The site-specific binding of bis-HT(I)-7(NMe) with dsDNA was demonstrated using the footprinting approach.
The work was supported by the RFBR (07-03-00492, 05-04-48743); the Program of Presidium of RAS on Molecular and Cell Biology; and the State program ?Design of Medicinal Products for Treatment and Prevention of Virus-Induced Diseases Using Chemical Synthesis.?
References and Footnotes
A. L. Zhuze1
1Engelhardt Institute of Molecular Biology