Sequence-specific alkylation by Y-shaped and tandem hairpin pyrrole-imidazole polyamides

To extend the target DNA sequence length of the hairpin pyrrole-imidazole (Py-Im) polyamide 1, we designed and synthesized Y-shaped and tandem hairpin Py-Im polyamides 2 and 3, which possess 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) as DNA-alkylating moieties. High-resolution denaturing polyacrylamide gel electrophoresis by using 5'-Texas-Red-labeled 465 base pair (bp) DNA fragments revealed that conjugates 2 and 3 alkylated the adenine of the target DNA sequences at nanomolar concentrations. Conjugate 2 alkylated adenine N3 at the 3' end of two 8 bp match sequences, 5'-AATAACCA-3' (site A) and 5'-AAATTCCA-3' (site C), while conjugate 3 recognized one 10 bp match sequence, 5'-AGAATAACCA-3' (site A) in the 465 bp DNA fragments. These results demonstrate that seco-CBI conjugates of Y-shaped and tandem hairpin polyamides have extended their target alkylation sequences.     

Sequence-specific gene silencing in mammalian cells by alkylating pyrrole-imidazole polyamides

Gene silencing was examined by sequence-specific alkylation of DNA by N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides. Polyamides ImImPyPygammaImImPyLDu86 (A) and ImImPyPygammaImPyPyLDu86 (B) selectively alkylated the coding regions of the renilla and firefly luciferases, respectively, according to the base pair recognition rule of Py-Im polyamides. Two different plasmids, encoding renilla luciferase and firefly luciferase, were used as vectors to examine the effect of alkylation on gene silencing. Transfection of the alkylated luciferase vectors-by polyamide A or B-into HeLa, 293, and NIH3T3 cells demonstrated that these sequence-specific DNA alkylations lead to selective silencing of gene expression. Next, the vectors were cotransfected into HeLa cells and the cells were treated with polyamide A or B. Selective reduction of luciferase activities was caused by both polyamides. On the basis of this sequence-specific alkylation and gene silencing activity, these alkylating Py-Im polyamides thus have potential as antitumor drugs to target specific gene expression in human cells.     

Sequence-specific alkylation of double-strand human telomere repeat sequence by pyrrole-imidazole polyamides with indole linkers

We designed and synthesized pyrrole (Py)-imidazole (Im) hairpin polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates 1 and 2, which target both strands of the double-stranded region of the human telomere repeat sequences, 5'-d(TTAGGG)(n)-3'/5'-d(CCCTAA)(n)-3'. High-resolution denaturing polyacrylamide gel electrophoresis demonstrated that conjugates 1 and 2 alkylated DNA at the 3' A of 5'-ACCCTA-3' and 5'-AGGGTTA-3', respectively. Cytotoxicities of conjugates 1 and 2 were evaluated using 39 human cancer cell lines; averages of log IC(50) values for conjugates 1 and 2 were -6.96 (110 nM) and -7.24 (57.5 nM), respectively. Conjugates 1 and 2 have potential as antitumor drugs capable of targeting telomere repeat sequence.     

DNA alkylation by pyrrole-imidazole seco-CBI conjugates with an indole linker: sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation

The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.     

Sequence-selective single-molecule alkylation with a pyrrole-imidazole polyamide visualized in a DNA nanoscaffold

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (～200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.     

Enantioselective DNA alkylation by a pyrrole-imidazole S-CBI conjugate

Conjugates 12S and 12R of N-methylpyrrole (Py)-N-methylimidazole (Im) seven-ringed hairpin polyamide with both enantiomers of 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) were synthesized, and their DNA alkylating activity was examined. High-resolution denaturing gel electrophoresis revealed that 12S selectively and efficiently alkylated at one match sequence, 5'-TGACCA-3', in 450-bp DNA fragments. The selectivity and efficiency of the DNA alkylation by 12S were higher than those of the corresponding cyclopropapyrroloindole (CPI) conjugate, 11. In sharp contrast, another enantiomer, 12R, showed very weak DNA alkylating activity. Product analysis of the synthetic decanucleotide confirmed that the alkylating activity of 12S was comparable with 11 and that 12S had a significantly higher reactivity than 12R. The enantioselective reactivity of 12S and 12R is assumed to be due to the location of the alkylating cyclopropane ring of the CBI unit in the minor groove of the DNA duplex. Since the CBI unit can be synthesized from commercially available 1,3-naphthalenediol, the present results open up the possibility of large-scale synthesis of alkylating Py-Im polyamides for facilitating their use in future animal studies.     

Sequence‐Specific DNA Alkylation Targeting for Kras Codon 13 Mutation by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Hairpin N‐methylpyrrole‐N‐methylimidazole polyamide seco‐CBI conjugates 2 – 6 were designed for synthesis by Fmoc solid‐phase synthesis, and their DNA‐alkylating activities against the Kras codon 13 mutation were compared by high‐resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5′‐ACGTCACC A ‐3′ (site 2), including minor 1 bp‐mismatch alkylation against wild type 5′‐ACG C CACC A ‐3′ (site 3). Conjugate 6 , which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5′‐ACGTCACC A ‐3′ (site 2). The hairpin polyamide seco‐CBI conjugate 5 thus alkylates according to Dervan′s pairing rule with the pairing recognition which β/β pair targets T–A and A–T pairs. SPR and a computer‐minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.     

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5'-AGTCAG-3' (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5'-AGTCAG-3' (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5'-AGTCCA-3' (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5'-TCACAA-3' (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervan's pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.     

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.     

Sequence-specific trapping of topoisomerase I by DNA binding polyamide-camptothecin conjugates

Hairpin pyrrole-imidazole polyamides are synthetic ligands that bind in the minor groove of DNA with affinities and specificities comparable to those of DNA binding proteins. Three polyamide-camptothecin conjugates 1-3 with linkers varying in length between 7, 13, and 18 atoms were synthesized to trap the enzyme Topoisomerase I and induce cleavage at predetermined DNA sites. One of these, polyamide-camptothecin conjugate 3 at nanomolar concentration (50 nM) in the presence of Topo I (37 degrees C), induces DNA cleavage between three and four base pairs from the polyamide binding site in high yield (77%).     

Programmable pyrrole-imidazole polyamides: A potent tool for DNA targeting

Pyrrole-imidazole (Py-Im) polyamides are a class of programmable minor-groove binders that recognize pre-determined DNA double helixes with high affinity and specificity. This review summarized the recent advances of Py-Im polyamides from their synthesis to applications via various modifications at the molecular level.     

Photoresponsive DNA Nanocapsule Having an Open/Close System for Capture and Release of Nanomaterials

A photofunctionalized square bipyramidal DNA nanocapsule (NC) was designed and prepared for the creation of a nanomaterial carrier. Photocontrollable open/close system and toehold system were introduced into the NC for the inclusion and release of a gold nanoparticle (AuNP) by photoirradiation and strand displacement. The reversible open and closed states were examined by gel electrophoresis and atomic force microscopy (AFM), and the open behavior was directly observed by high‐speed AFM. The encapsulation of the DNA‐modified AuNP within the NC was carried out by hybridization of a specific DNA strand (capture strand), and the release of the AuNP was examined by addition of toehold‐containing complementary DNA strand (release strand). The release of the AuNP from the NC was achieved by the opening of the NC and subsequent strand displacement.     

Pharmacokinetics of Py-Im polyamides depend on architecture: cyclic versus linear

The pharmacokinetic properties of three pyrrole-imidazole (Py-Im) polyamides of similar size and Py-Im content but different shape were studied in the mouse. Remarkably, hairpin and cyclic oligomers programmed for the same DNA sequence 5'-WGGWWW-3' displayed distinct pharmacokinetic properties. Furthermore, the hairpin 1 and cycle 2 exhibited vastly different animal toxicities. These data provide a foundation for design of DNA binding Py-Im polyamides to be tested in vivo.     

C-H to N substitution dramatically alters the sequence-specific DNA alkylation, cytotoxicity, and expression of human cancer cell lines

We designed and synthesized sequence-specific alkylating conjugates 1 and 2, which selectively alkylate matched sequences at nanomolar concentrations. Conjugates 1 and 2 differ only in that the C-H is substituted by an N in the second ring, which precisely recognizes and effectively alkylates DNA according to the recognition rule of Py-Im polyamides. We investigated sequence-specific DNA alkylation, cytotoxicity in 39 human cancer cell lines, and the effect on expression levels in cancer cell lines by Py-Im conjugates 1 and 2. The COMPARE analysis of the mean graphs showed that conjugates 1 and 2 did not correlate well with each other (r = 0.65) despite having a common DNA alkylating mechanism (purine N3 alkylation). Array-based gene expression analysis demonstrated that there are several oppositely regulated genes. The results suggest the intriguing possibility that DNA alkylating agents recognizing longer base-pair sequences may provide a promising approach for developing new types of antigene agents.     

Only complete rejoining of DNA strand breaks after UVC allows K562 cell proliferation and DMSO induction of erythropoiesis

DNA strand breaks are early intermediates of the repair of UVC-induced DNA damage, however, since they severely impair cellular activities, their presence should be limited in time. In this study, the effects of incomplete repair of UVC-induced DNA strand breaks are investigated on K562 cell growth and the induction of erythroid differentiation by addition of DMSO to the cell culture medium. The kinetics were followed after UV irradiation by single cell gel electrophoresis, and in total cell population by alkaline or neutral agarose gel electrophoresis. Shortly after exposure, an extensive fragmentation occurred in DNA; DNA double strand breaks were negatively correlated with recovery time for DNA integrity. DNA damage induced by UVC 9J/m2 rapidly triggered necrosis in a large fraction of irradiated K562 cells, and only 40% of treated cells resumed growth at a very low rate within 24h of culture. The addition of DMSO to the culture medium of cells 15min after UVC, when DNA strand break repair was not yet complete, produced apoptosis in >70% of surviving cells, as determined by TUNEL assay. Conversely, if DMSO was added when the resealing of DNA strand breaks was complete, surviving K562 cells retained full growth capacity, and their progeny underwent erythroid differentiation with normal levels of erythroid proteins, delta-aminolevulinic acid dehydrase and hemoglobin. This study shows that the extent of DNA strand break repair influences cell proliferation and the DMSO induced erythroid program, and the same UVC dose can have opposite effects depending on cellular status.     

Determination of Adenosine Triphosphate by a Target Inhibited Catalytic Cycle Based on a Strand Displacement Reaction

A strand displacement reaction-based system was developed for the determination of adenosine triphosphate (ATP). It involved an entropy-driven catalytic cycle that directly employed the ATP aptamer as the catalyst. Introduction of ATP into the system induced the catalyst to form the G-quadruplex conformation and inhibited its catalytic activity. All intermediates in the catalytic cycle processes were identified by polyacrylamide gel electrophoresis analysis. When the oligonucleotides were labeled with a carboxyfluorescein fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid quencher, this strand displacement reaction-based catalytic system exhibited a “switch-on” response for ATP. Conditions for detecting ATP, such as the toehold length, concentrations of the catalyst and magnesium ion, and incubation temperature, were optimized to obtain a detection limit of 50 nM and a linear response up to 1400 nM of ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing.     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Deoxyribonucleic acid strand break induced by the hydroxy radical-generating cyclic peroxide, 4-ethoxy-1,4-dihydro-2,3-benzodioxin-1-ol.

Deoxyribonucleic acid (DNA) strand break caused by a synthetic cyclic peroxide, 4-ethoxy-1,4-dihydro-2,3-benzodioxin-1-ol (Bd) was studied by both ethidium bromide fluorescence quenching and agarose gel electrophoresis. The Bd-mediated DNA strand break occurred dependently on temperature and also on Bd concentration. The reaction proceeded at a temperature higher than 30 degrees C (decomposition temperature of Bd), indicating that the reactive species generated by Bd-decomposition are responsible for the reaction. The reaction was protected by 1,4-diazabicyclo[2.2.2]octane and NaN3, and also moderately by OH radical scavengers such as Na-benzoate and NaBr. Possible involvement of active oxygen radicals including OH radical in the DNA strand break is discussed in relation to the mechanism of Bd decomposition.     

Effects of sequence and structure in the separation of nucleic acids using ion pair reverse phase liquid chromatography

Ion pair reverse phase high performance liquid chromatography on non-porous alkylated poly(styrene-divinylbenzene) particles enables the high resolution separation of double stranded DNA fragments. To further understand the separation mechanisms involved in ion pair reverse phase liquid chromatography we have analysed the effects of curved or "bent" DNA fragments with respect to their separation using both gel electrophoresis and ion pair reverse phase liquid chromatography. Size dependent separations of curved DNA fragments that migrate anomalously during gel electrophoresis were observed using ion pair reverse phase liquid chromatography. To further study the sequence effect and resulting changes in hydrophobicity of the duplex DNA, PCR fragments were generated that contain uracil in place of thymine. The resulting fragments were shown to elute with shorter retention times, demonstrating that sequence-specific effects can alter the retention of duplex DNA. The study was extended to the investigation of non-canonical B-DNA structures (Holliday junctions) under various chromatographic conditions, demonstrating that the coaxial stacking of the helices in such structures, in the presence of magnesium causes a change in retention.