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.     

Fluorescence detection of cytosine/guanine transversion based on a hydrogen bond forming ligand

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate potential use of a hydrogen bond forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), for the fluorescence detection of the cytosine (C)/guanine (G) mutation sequence of the cancer repression gene p53. Our method is based on construction of the AP site in ODN duplexes, which allows small synthetic ligands to bind to target nucleobases accompanied by fluorescence signaling: an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. In 10 mM sodium cacodylate buffer solutions (pH, 7.0) containing 100 mM NaCl and 1.0 mM EDTA, AMND is found to strongly bind to C (K_d=1.5×10⁻⁶ M) in the target ODN while the binding affinity for G is relatively moderate (K_d=50×10⁻⁶ M). Significant fluorescence quenching of AMND is observed only when binding to C, making it possible to judge the C/G transversion with the naked eye.     

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag⁺ binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag⁺ preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag⁺ binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.     

基于核酸脱碱基位点的铽离子荧光增强型单核苷酸多态性识别研究

基于核酸脱碱基(AP)位点构建了无机配体稀土铽离子(Tb^3＋)荧光增强型单核苷酸多态性(SNP)识别方法. 在目标链靶标碱基对应的探针链上相应位置引入AP位点, 发现Tb^3＋可以选择性地结合在AP位点, 光激发时发生从DNA碱基到结合的Tb^3＋的能量转移, 使Tb^3＋特征荧光显著增强. 这种荧光增强作用与靶标碱基及AP位点侧翼碱基类型密切相关. 当靶标碱基和侧翼碱基为G时, 荧光最强. 该方法可用于区分肿瘤抑制基因p53密码子177位的C/G碱基变异.     

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.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

Use of vitamin B2 for fluorescence detection of thymidine-related single-nucleotide polymorphisms

In combination with abasic site (AP site)-containing DNAs, potential use of a biotic fluorescence compound, Vitamin B₂ (riboflavin), is demonstrated for the fluorescence detection of the thymine (T)-related single-nucleotide polymorphisms. Our method is based on construction of the AP site in DNA duplexes, which allows small ligands to bind to target nucleotides accompanied by fluorescence signaling: an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleotides through stacking and hydrogen-bonding interactions. In 10 mM sodium cacodylate buffer solutions (pH 7.0) containing 100 mM NaCl and 1.0 mM EDTA, Vitamin B₂ is found to selectively bind to T (K₁₁ = 1.8 × 10⁶ M⁻¹ at 5 °C) over other nucleobases, and this is accompanied by significant quenching of its fluorescence. While the sensing functions depend on the flanking sequences to the AP site, Vitamin B₂ is applicable to the detection of T/C (cytosine), T/G (guanine) and T/A (adenine) mutation sequences of the CYP2A6 gene, where the flanking nucleobases are guanines in both positions (-GXG-, X = AP site).     

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.     

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.     

Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10⁹ nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

Fluorescence light-up recognition of DNA nucleotide based on selective abasic site binding of an excited-state intramolecular proton transfer probe

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.     

Formation of an amino-acid-binding pocket through adaptive zippering-up of a large DNA hairpin loop

Background: In vitro selection has identified DNA aptamers that target cofactors, amino acids, peptides and proteins. Structure determination of such ligand-DNA aptamer complexes should elucidate the details of adaptive DNA structural transitions, binding-pocket architectures and ligand recognition. We have determined the solution structure of the complex of a DNA aptamer containing a guanine-rich 18-residue hairpin loop that binds l-argininamide with ∼ 100μM affinity.Results: The DNA aptamer generates its l-argininamide-binding pocket by adaptive zippering up the 18-residue loop through formation of Watson-Crick pairs, mismatch pairs and base triples, while maximizing stacking interactions. Three of the four base triples involve minor-groove recognition through sheared G·A mismatch formation. The unique fold is also achieved through positioning of an adenine residue deep within the minor groove and through nestling of a smaller loop within the larger loop on complex formation. The accessibility to the unique l-argininamide-binding pocket is restricted by a base pair that bridges across one side of the major-groove-binding site. The guanidinium group of the bound l-argininamide aligns through intermolecular hydrogen-bond formation with the base edges of nonadjacent guanine and cytosine residues while being sandwiched between the planes of nonadjacent guanine residues.Conclusions: The available structures of l-arginine/l-argininamide bound to their DNA and RNA targets define the common principles and patterns associated with molecular recognition, as well as the diversity of intermolecular hydrogen-bonding alignments associated with the distinct binding pockets.     

Aromatic interaction profile to understand the molecular basis of raltegravir resistance

Integrase (IN) is the enzyme of human immunodeficiency virus (HIV) which inserts the viral DNA (vDNA) into the host genome for successful viral replication leading to the infection. However, the chemical basis of HIV IN catalysis is speculative due to lack of complete co-crystal structure. Using the recently published prototype foamy virus IN crystal structure, we developed a model structure of HIV IN showing interaction of vDNA, the metal (Mg²⁺) cofactor, and raltegravir (RLT) in the active site. Molecular docking and dynamics simulations studies showed that RLT uses it core central ring with diketo motif for Mg²⁺ chelation and bridge interaction with DDE motif. The triple arene interactions mediated by RLT with neighboring molecular motifs (Y143, cytosine, and adenine) is maintained during long simulation in wild type (WT). The fluorobenzyl and oxadiazole moieties of RLT forms aromatic stacking with cytosine base (head stacking) aromatic side chain of Y143 (tail stacking), respectively, while central ring further establishes aromatic stacking with distorted adenine base of vDNA (central stacking). The novel triple stacking systems were further explored to understand the molecular basis of drug resistance by molecular simulation. The in silico mutation (N155H, Q148H, and Q148H + G140S) and simulation studies elucidated the structural mechanism of resistance to RLT. The simulation studies provided the molecular basis for interdependency observed for the primary and secondary (Q148H and G140S) mutations and also explained the mechanism of viral fitness regain. Our study reveals that triple stacking and its consequence in terms of VdW energetic profile acts as a critical point to understand the drug-resistance. Here, we demonstrate that the root mean square deviation of centroid system (aromatic stacking) can be used as a major determinant of RLT binding toward the fold resistance. This is first kind of report, which discloses a strategy to explore the molecular level of drug resistance profile using aromatic interactions.     

带电组氨酸侧链与DNA碱基间非键作用强度的理论研究

采用MP2方法和6-31+G(d,p)基组优化得到了带有一个正电荷的组氨酸侧链与4个DNA碱基间形成的18个氢键复合物的气相稳定结构, 从文献中获取了组氨酸侧链与DNA碱基间形成的12个堆积和T型复合物的气相稳定结构, 使用包含基组重叠误差(BSSE)校正的MP2方法和aug-cc-pVTZ基组及密度泛函理论M06-2X-D3方法和aug-cc-pVDZ基组计算了这些复合物的结合能. 研究结果表明, 包含BSSE校正的M06-2X-D3方法和aug-cc-pVDZ基组能够给出较准确的结合能; 气相条件下, 组氨酸侧链与同种DNA碱基间的离子氢键作用明显强于堆积作用和T型作用, 组氨酸侧链最易通过离子氢键与胞嘧啶C和鸟嘌呤G作用形成氢键复合物, 组氨酸与胞嘧啶C和鸟嘌呤G间的T型作用强于与腺嘌呤A和胸腺嘧啶T间的离子氢键作用; 水相条件下, 组氨酸侧链与同种DNA碱基间的离子氢键作用仍明显强于堆积作用和T型作用, 组氨酸侧链更易与胞嘧啶C和鸟嘌呤G相互作用形成氢键复合物, 但是最强的组氨酸侧链与胞嘧啶C间的T型作用明显弱于与腺嘌呤A和胸腺嘧啶T间的离子氢键作用, 说明水相条件下组氨酸侧链与DNA碱基间主要通过离子氢键作用形成氢键复合物.     

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.     

Direct electrochemistry and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.     

Enantiodivergent formation of a chiral cytosine crystal by removal of crystal water from an achiral monohydrate crystal under reduced pressure

An achiral nucleobase cytosine forms an achiral monohydrate crystal (space group: P2₁/c) by crystallization from a water solution. It was found that the removal of crystal water under reduced pressure at room temperature afforded a chiral crystal of anhydrous cytosine (P2₁2₁2₁). The crystal chirality of anhydrous cytosine corresponds to the enantiotopic crystal face of the achiral monohydrate; therefore, when the enantiotopic b¹-face is exposed to the reduced pressure, dehydration occurred in the direction from the b¹-face to provide [CD(+)310_KBr]-cytosine crystal. In contrast, dehydration from the b²-face gave the opposite enantiomorphous [CD(?)310_KBr]-cytosine crystal. The correlation between enantiotopic faces and the formed crystal chirality is opposite to that from dehydration by heating. The formed chiral cytosine crystals act as a chiral trigger for asymmetric autocatalysis with enantioenrichment amplification of pyrimidylalkanol.     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

Strong and selective binding of amiloride to an abasic site in RNA duplexes: thermodynamic characterization and microRNA detection

Firmly tied: The binding affinity of amiloride for an abasic (AP) site-containing RNA duplex is two orders of magnitude superior to the affinity of the corresponding AP site-containing DNA duplex. The observed high binding affinity for the RNA duplex arises from a favorable enthalpy gain. The binding-induced fluorescence response of amiloride is applicable to microRNA detection.