New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies.     

Fluorescence and electrochemical detection of pyrimidine/purine transversion by a ferrocenyl aminonaphthyridine derivative

A novel hydrogen bond-forming ligand for pyrimidine/purine transversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.     

Synthesis and analysis of oligonucleotides containing abasic site analogues

DNA damage results in the formation of abasic sites from the formal hydrolysis of the glycosidic bond (AP) and several oxidized abasic lesions. Previous studies on AP sites revealed that DNA polymerases preferentially incorporated dA opposite them in approximately 80% of the replication events in Escherichia coli. These results were consistent with the hypothesis that the AP sites are noninstructive lesions due to the absence of a Watson-Crick base whose bypass adheres to the "A-rule." Recent replication studies of the oxidized abasic lesion, 2-deoxyribonolactone (L), revealed that DNA polymerase(s) does not apply the A-rule when bypassing it and incorporates large amounts of dG opposite L. These studies suggested that abasic sites such as L do direct polymerases to selectively incorporate nucleotides opposite them. However, it was not possible to determine the structural basis for this molecular recognition from these experiments. A group of oligonucleotides containing analogues of the AP and L lesions were synthesized and characterized as probes to gain insight into the structural basis for the distinct effect of 2-deoxyribonolactone on replication. These molecules will be useful tools for studying replication in cells and in vitro.     

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.     

Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.     

Enhancement of the binding ability of a ligand for nucleobase recognition by introducing a methyl group.

The recognition ability of pteridine derivatives for nucleobases opposite an abasic (AP) site in an oligodeoxynucleotide (ODN) duplex is enhanced by using a propylene residue (Spacer-C3) as an AP site. The recognition ability is further enhanced both by attaching methyl groups to a fluorescent ligand and by measuring the fluorescence response at 5 degrees C; 6.2 x 10(6) M(-1) of the binding constant is attained between 2-amino-6,7-dimethyl-4-hydroxypteridine and guanine opposite the AP site in water.     

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a_n′ and w_n′) produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w_n′ ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an “in situ” derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5′-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3′ and 5′ fragments (w_n−m, b_m, a_m, d_m−1) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site.     

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 °C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.     

Use of abasic site-containing DNA strands for nucleobase recognition in water

Nucleobase recognition in water is successfully achieved by the use of an abasic site (AP site) as the molecular recognition field. We intentionally construct the AP site in DNA duplex so as to orient the AP site toward a target nucleobase and examine the complexation of 2-amino-7-methylnaphthyridine (AMND) with nucleobases at the AP site. AMND is found to selectively bind to cytosine (C) base with a 1:1 binding constant of >106 M-1, accompanied by remarkable quenching of its fluorescence. In addition to hydrogen bonding, a stacking interaction with nucleobases flanking the AP site seems responsible for the binding properties of AMND at the AP site. Possible use of AMND is also presented for selective and visible detection of a single-base alternation related to the cytosine base.     

Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA

Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin–DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3′-end and an oligonucleotide with a phosphorylated 5′-end.     

Photochemical generation of ribose abasic sites in RNA oligonucleotides

[reaction: see text] A general method for the photochemical generation of ribose abasic sites within RNA oligonucleotides is reported. Photochemically caged nucleoside phosphoramidite analogues were prepared and incorporated into RNA oligonucleotides by automated RNA synthesis. Irradiation of the modified RNA at 350 nm efficiently produced ribose abasic sites at specific sites within RNA sequences. The current approach offers a chemical route to RNA abasic lesions for RNA biochemical studies.     

Synthesis of purine-acridine hybrid molecules related to artificial endonucleases

In the course of a program devoted to the synthesis of artificial endonucleases, we have previously reported a series of hybrid molecules in which a purine is linked to an intercalating drug by a polyamino chain. These molecules recognize and cleave selectively abasic sites in DNA with very high efficiency. In order to get insight into the mechanism of recognition and cleavage, we have prepared a new series of molecules in which the purine is linked to an amino-acridine by an aliphatic chain containing amido or/and amino groups. The key intermediates are -halo-ω-amino polyaza which may be of general use as linkers in bioconjugate chemistry.     

Synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in DNA

[structure: see text] The synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in damaged nucleic acids is described. The reagent reacts readily with abasic sites of damaged calf thymus DNA generated in a time-dependent manner under acid/heat depurination conditions. Preliminary results indicate the sensitivity of the direct chemiluminescent detection format is approximately 0.1 abasic sites detected per 10(6) nucleotides using as little as 200 ng of DNA.     

Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy

A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a single fluorochrome-DNA complex was calibrated. The number of abasic sites on the DNA molecule was estimated by counting each fluorochrome-DNA complex. The present study directly visualized and characterized the abasic sites of single DNA molecules.     

Nucleic acid structural engineering using pyrene-functionalized 2'-amino-alpha-L-LNA monomers and abasic sites

Oligonucleotides (ONs) modified with a 2'-N-(pyren-1-yl)acetyl-2'-amino-alpha-L-LNA thymine monomer Y flanked on the 3'-side by an abasic site Phi (i.e., YPhi-unit) exhibit unprecedented increases in thermal affinity (DeltaT(m) values) toward target strands containing abasic sites (DeltaT(m) per YPhi unit >+33.0 degrees C in 9-mer duplexes relative to unmodified ONs). Biophysical studies along with force field calculations suggest that the conformationally locked 2-oxo-5-azabicyclo[2.2.1]heptane skeleton of monomer Y, in concert with the short rigid acetyl linker, efficiently forces the thymine and pyrene moieties to adopt an interplanar distance of approximately 3.4 A. This precisely positions the pyrene moiety in the duplex core void formed by abasic sites (Phi:Phi pair) for optimal pi-pi overlap. Duplexes with multiple YPhi: APhi units separated by one base pair are tolerated extraordinarily well, as exemplified by a 13-mer duplex containing four separated YPhi: APhi units (8 abasic sites distributed over 13 "base pairs"), which exhibit a thermal denaturation temperature of 60.5 degrees C. The YPhi probes display up to 16-fold increases in fluorescence intensity at 380 nm upon hybridization with abasic target strands, whereby self-assembly of these complex architectures can be easily monitored. This study underlines the potential of N2'-functionalized 2'-amino-alpha-L-LNA as building blocks in nucleic acid based diagnostics and nanomaterial engineering.     

Molecular modeling study of DNA abasic sites

We use molecular modeling calculations to study the structure and the flexibility of abasic (AP sites) and for the design of anticancer drugs targeted against AP sites. For either adenine or cytosine on the opposing strand within the same sequence context, the results are in line with experimental data which show that the two unpaired bases lead to intrahelical forms, but with differences in induced curvature. Results on flexibility, indicate that the two duplexes have the same bending rigidity for cytosine. In previous work a series of polyfunctional molecules, such as ATAc, were designed to selectively recognize and cleave abasic sites in DNA. The nitrobenzamide group which was added to the ATAc molecule to obtain a new molecule, termed ATAc4, can induce a second lesion under irradiation in close proximity to the abasic site. The different conformations of ATAc4 interacting with a DNA oligomer containing a stable analog of the abasic site were compared to the photoinduced cleavage pattern observed experimentally. Received: 16 September 1999 / Accepted: 3 February 2000 / Published online: 12 May 2000     

Luminescence-based colorimetric discrimination of single-nucleotide transversions by the combined use of the derivatives of DOTA-conjugated naphthyridine and its terbium complex

We newly synthesized a nucleobase-binding ligand, ND-DOTA, in which 2-amino-5,7-dimethyl-1,8-naphthyridine (ND) was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DOTA) via an amide linker, and found that its terbium(III) complex (ND-DOTA-Tb) showed green emission based on an energy transfer from the naphthyridine moiety to Tb³⁺. The blue emission of ND-DOTA was selectively quenched by adding abasic site-containing DNA duplexes that have pyrimidine bases opposite to the abasic site. In contrast, at the same excitation wavelength, ND-DOTA-Tb showed green emission independently of the bases opposite to the abasic site. Thus, a mixed solution of ND-DOTA and ND-DOTA-Tb enabled the luminescence-based colorimetric discrimination of single-nucleotide transversions with the naked eye at a single excitation wavelength.     

Generation of an abasic site in an oligonucleotide by using acid-labile 1-deaza-2'-deoxyguanosine and its application to postsynthetic modification

We developed a new convenient method for generation of an abasic site at the 3'-terminus of an oligonucleotide. This method uses a 1-deaza-2'-deoxyguanosine residue, which easily undergoes depurination under acidic conditions. The abasic site of the oligonucleotide can be further modified with external functional groups. We report herein the chemical stability of 1-deaza-2'-deoxyguanosine in the oligodeoxynucleotide and the application to the postsynthetic modification of an oligonucleotide by utilizing the chemical property of 1-deaza-2'-deoxyguanosine. [Structure: see text]     

An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis

Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.     

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.