Chemically induced hairpin formation in DNA monolayers

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.     

Induction of a remarkable conformational change in a human telomeric sequence by the binding of naphthyridine dimer: inhibition of the elongation of a telomeric repeat by telomerase

The binding of a dimeric form of the 2-amino-1,8-naphthyridine derivative (naphthyridine dimer) to a human telomeric sequence, TTAGGG, was investigated by UV melting, CD spectra, and CSI-MS measurements. Both the 9-mer d(TTAGGGTTA) and the 15-mer d(TTAGGGTTAGGGTTA) showed apparent melting temperatures (T(m)) of 45.6 and 63.6 degrees C, respectively, in the presence of naphthyridine dimer (30 microM) in sodium cacodylate buffer (50 mM, pH 7.0) containing 100 mM NaCl. The CD spectra at 235 and 255 nm of the 9-mer increased in intensity accompanied with strong induced CDs at 285 and 340 nm upon complex formation with naphthyridine dimer. UV titration of the binding of naphthyridine dimer to the 9-mer at 320 nm showed a hypochromism of the spectra. A Scatchard plot of the data showed the presence of multiple binding sites with different association constants. Cold spray ionization mass spectrometry of the complex between naphthyridine dimer and the 9-mer clearly showed that one to three molecules of the ligand bound to the dimer duplex of the 9-mer. Telomeric repeat elongation assay showed that the binding of naphthyridine dimer to the telomeric sequence inhibits the elongation of the sequence by telomerase.     

Insertion of a bulky rhodium complex into a DNA cytosine-cytosine mismatch: an NMR solution study

The bulky octahedral complex Rh(bpy)2chrysi3+ (chrysi = 5,6-chrysenequinonediimine) binds single-base mismatches in a DNA duplex with micromolar binding affinities and high selectivity. Here we present an NMR solution study to characterize the binding mode of this bulky metal complex with its target CC mismatch in the oligonucleotide duplex (5'-CGGACTCCG-3')2. Both NOESY and COSY studies indicate that Rh(bpy)2chrysi3+ inserts deeply in the DNA at the mismatch site via the minor groove and with ejection of both destabilized cytosines into the opposite major groove. The insertion only minimally distorts the conformation of the oligonucleotide local to the binding site. Both flanking, well-matched base pairs remain tightly hydrogen-bonded to each other, and 2D DQF-COSY experiments indicate that all sugars maintain their original C2'-endo conformation. Remarkably, 31P NMR reveals that opening of the phosphate angles from a BI to a BII conformation is sufficient for insertion of the bulky metal complex. These results corroborate those obtained crystallographically and, importantly, provide structural evidence for this specific insertion mode in solution.     

Targeting abasic sites and single base bulges in DNA with metalloinsertors

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.     

Affinity labeling of a single guanine bulge

We have developed a conceptually new method for the selective labeling of duplex DNA containing a guanine bulge with a masked form of fluorescent 2-amino-1,8-naphthyridine. A naphthyridine derivative 2 tethering DNA-alkylating epoxide was synthesized from (S)-epichlorohydrin and naphthyridine derivative 1, which selectively binds to the guanine bulge duplex. HPLC analysis of the labeling reaction of bulge duplex d(GTT GTGTTG GA)/d(CAA CA A ACC T) (TGT/A_A) with 2 showed a formation of 2-TGT adduct for the guanine bulge. The reaction proceeded for the guanine bulge and a reduced efficiency for the cytosine bulge, but not at all for adenine and thymine bulges. The site of covalent bond formation in 2-TGT was unambiguously identified at the guanine two bases away from the bulge by the use of MALDI-TOF MS analysis of the oligomer fragments produced by strand scission. The labeling reaction was also observed for the guanine bulge flanking two G-C base pairs (CGC/G_G), producing a 2:1 adduct (2.2-CGC). Upon hydrolysis of 2-TGT and 2.2-CGC with concentrated hydrogen chloride, a release of fluorescent 2-aminonaphthyridine from the adduct was clearly detected, verifying a concept of an affinity labeling of the guanine bulge with a masked fluorescent chromophore. The affinity labeling targeting of the guanine bulge is a conceptually novel method for the postsynthetic labeling of DNA. Hybridization, to the target sequence, of a probe DNA possessing one extra guanine especially between two cytosines provides a unique site for the labeling by masked fluorophore 2. The technique may have broad application in genetic typing without using a conventional synthesis of fluorescent-labeled DNA oligomers.     

Polyamine-Functionalized 2′-Amino-LNA in Oligonucleotides: Facile Synthesis of New Monomers and High-Affinity Binding towards ssDNA and dsDNA

Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2′-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2′-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson–Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3′-nucleolytic digestion relative to the non-functionalized controls.     

A facile route to bimetallic ruthenium dipyridophenazine complexes

Using achiral coordinatively unsaturated metal complex building-blocks, the two step synthesis of a bimetallic complex containing independent [Ru(II)dppz] units tethered together by a linking 4,4'dipyridyl-1,5-pentane ligand is reported. Photophysical studies on this prototype system indicate that the characteristic luminescence of the [Ru(II)dppz] moieties is perturbed by self-quenching processes. Preliminary binding studies on the complex with natural and synthetic duplex DNA is reported. Luminescence and calorimetric titrations reveal that the complex does not show enhanced binding affinity with respect to analogous monometallic complexes. This result is interpreted by a consideration of the length and rigidity of the linker employed in the complex.     

Influence of the Type of Junction in DNA-3′-Peptide Nucleic Acid (PNA) Chimeras on Their Binding Affinity to DNA and RNA

The automated on-line synthesis of DNA-3′-PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X=S), a N-(2-hydroxyethyl)- (X=O), or a N-(2-aminoethyl)- (X=NH) N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3′-PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.     

Intercalating nucleic acids (INAs) containing insertions of 6H-indolo[2,3-b]quinoxaline

6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the oligo and the intercalator must consist of at least five C atoms in order to stabilize a DNA duplex. An intercalator with a 2′-deoxy-d-riboside linker to the oligo could also stabilize a DNA/RNA duplex, while (S)-4-(6-methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butane-1,2-diol was able to stabilize both DNA/DNA, DNA/RNA and a DNA/LNA duplex. Mismatch studies revealed a huge sensitivity to the C-C mismatch at the 5′-site of the intercalator.     

二芳基三嗪类HIV-1逆转录酶抑制剂的分子对接及3D-QSAR研究

对40个二芳基三嗪类HIV-1逆转录酶抑制剂进行了分子对接研究,结果表明,2个疏水性芳香取代基团与结合口袋底部形成的疏水和范德华相互作用、三嗪环母核及其R4取代基与结合位点产生的氢键和静电作用以及R4取代基与袋口形成的空间位阻效应是影响该系列化合物活性的重要因素.根据对接优势构象进行分子叠合和比较分子相似性指数分析(C...     

Fluorescent Short-Stranded Helical Foldamers Based on L-shaped Dibenzopyrrolo[1,2-a][1,8]naphthyridine

Helical structures were constructed by using π-spacer-bridged dimers of dibenzopyrrolo[1,2-a][1,8]naphthyridine, which has a highly fluorescent L-shaped π-extended skeleton. Three dimers with biphenylene (dimer 1 ), phenanthrene (dimer 2 ), and m-phenylene (dimer 3 ) spacers, as well as a fixed-helical dimer 4 where two quinolone rings were covalently cross-linked, were designed and prepared. ¹H NMR and ROESY spectra revealed that dimers 1 and 2 adopted helical forms in solution, whereas dimer 3 did not. The helical conformation of 1 was strengthened by addition of either polar or nonpolar solvents to the chloroform solution, which suggested that π–π stacking was the main contributor to the stabilization of the helical structure. All of the dimers, including fixed-helical dimer 4 , emitted fluorescence with high quantum yields (ϕ=0.79–0.86).     

Inter- and intrastrand DNA crosslinks by 2-fluoro-substituted pyrrolobenzodiazepine dimers: stability, stereochemistry and drug orientation

A 2-fluoro-substituted pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer with a 1,4-di-n-propyl piperazine linker was studied with respect to its binding and crosslinking capability towards double-helical DNA targets. Duplex thermal stabilizations upon drug binding as measured by UV melting experiments suggest that two guanine bases separated by four AT base pairs constitute the favorable binding site for the PBD dimer. Large stabilizations were observed for the self-complementary duplex d(AACAATTGTT)(2) as well as for the non-self-complementary duplex d(AAGAATTGTT)·d(AACAATTCTT) with both guanines located on the same strand. Formation of interstrand and intrastrand crosslinks by the covalent binding of both PBD moieties of the dimer to the exocyclic 2-amino group of the two guanine bases within the duplex minor groove was confirmed by NMR structural studies. In both the symmetric and non-symmetric DNA-PBD adducts the newly created stereogenic center at C11 of the tricyclic PBD subunits favors an S configuration. Different orientations of the PBD aromatic A-ring with respect to the covalently modified guanine as observed in the non-symmetric complex are shown to result in characteristic changes of PBD H11 and H11a proton chemical shifts. Based on a compilation of available NMR data on various PBD complexes, these differences may be used as valuable probes for the identification of PBD orientational preferences in DNA-PBD adducts.     

Fluorescence studies on the interaction of ethidium bromide with duplex, triplex and quadruplex DNA structures

Under different conditions, oligonucleotides can form several alternative DNA structures such as duplex, triplex and quadruplex. All these structures can interact with ethidium bromide (EB) and make its fluorescence intensity change. The fluorescence spectra and other related parameters provided by static fluorescence techniques showed that the interaction mechanisms between EB and these structures were not always the same. Among them, B type duplex and triplex DNA adopt an intercalative mode when binding to the EB, which has a relatively high efficiency of energy transfer and the fluorescence of EB cannot be quenched easily. While for the parallel duplex DNA, the interaction mode is an outside binding in which energy transfer can hardly happen and its fluorescence intensity as well as Stern-Volmer constant is almost the same to the free EB. For the quadruplex, the binding mechanism to EB is more complex. Results from the energy transfer and quenching studies indicate that the two interaction modes note     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

[structure: see text] The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1 with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H (1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs can be selectively recognized by the newly designed bidentate ligands.     

Ligand self-recognition in the stereoselective assembly of [2 + 2] metallomacrocycles from racemic chiral bisbipyridyl molecular clefts and zinc(II)

The synthesis of racemic and optically pure ligand L, in which two 6,6'-disubstituted bipyridines are connected by methyleneoxy linkers to the molecular cleft dibenzobicyclo[b,f][3.3.1]nona-5a,6a-diene-6,12-dione, is reported. In the presence of 2 equivalents of zinc(ii) trifluoromethansulfonate (+/-)- undergoes slow reversible coordination over 24 h to form a pair of enantiomeric [2 + 2] metallomacrocycles, [Zn2(+)L2](OTf)(4) and [Zn2(-)L2](OTf)(4) respectively, that contain either two (+)-L ligands or two (-)-L ligands. This assignment was confirmed by independent studies with either (+)-L or (-)-L which formed the same complexes but at a significantly faster rate (3 h), and circular dichroism spectra of [Zn2(+)L2](OTf)(4) and [Zn2(-)L2](OTf)(4) which gave signals of the same intensity with the opposite sign. Treatment of (+/-)-L or optically pure L with copper(I) showed rapid formation of a mixture of oligomers as well as the [2 + 2] metallomacrocycle. The complex Zn2L2(OTf)(4) exhibits slow exchange between two species on the NMR time scale at room temperature. The results are consistent with the formation of a library of metal complexes in which the zinc(ii) binds initially to the most accessible bipyridyl binding sites in (+/-)-. Equilibration over several hours results in self-recognition of enantiomeric ligands to form a pair of enantiomeric metallomacrocycles, which have been tentatively assigned as having the helical configuration. Slow exchange is attributed to the preference for both metal centres to adopt 6-coordinate geometries involving the linker oxygens, but are limited to exchanging 5-coordinate complexes due to the shape of the cleft and the short linker.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu(2+). Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu(2+), duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu(2+) ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu(2+) of the 1:2 Cu(2+):Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ(2)] moieties that are similar to the corresponding square planar synthetic complex formed between Cu(2+) and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal l-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.     

Molecular modeling on recognition of sheared and normal DNA by novel metal complex Λ- and Δ-[Co(phen)2hpip]

Recognition of sheared and normal DNA by a novel metal complex [Co(phen)₂hpip]³⁺ (phen=1,10-phenanthroline, HPIP=2-(2-hydroxyphenyl)imidazole[4,5-f][1,10]phenanethroline) is studied by molecular modeling. Calculating results indicate that, this complex can specifically recognize DNA segment of sequence –MMNNMM– (M means mismatch base pairs and N means normal base pairs). Intercalating from minor groove between the middle normal duplex into the sheared DNA with the depth of 1.2 nm is of preference and enantioselectivity is observed. Comparison on the two DNA structures of optimal conformation and analysis on the interaction between DNA and the two tail ligands of the complex show that, the effect of the two neighboring mismatch duplexes on the structure of the middle normal base pairs and the steric interaction between the mismatch duplexes and the two tail ligands of the complex are the essential reason to the segment specificity. Investigation on the detailed energy terms indicate that, in effecting enantioselectivity, the electrostatic distribution of the complex is in the majority and steric interaction is at the next place. But, steric interaction is surely the only factor determining the intercalating from minor groove.