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.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

A Competitive Strategy Coupled with Endonuclease‐Assisted Target Recycling for DNA Detection Using Silver‐Nanoparticle‐Tagged Carbon Nanospheres as Labels

A simple competitive strategy was designed for the sensitive detection of sequence‐specific DNA by combining endonuclease‐assisted target recycling and electrochemical stripping analysis of silver nanoparticles (AgNPs). The AgNP‐tagged carbon nanospheres were synthesized by means of in situ reduction of Ag⁺ adsorbed onto a negatively charged polyelectrolyte layer and functionalized with streptavidin for binding biotin‐labeled DNA strands. The labeled strand was captured on the DNA sensor surface by competitive hybridization of biotinated primer 1 and its cleaved product. The cleaved product could be amplified in homogeneous solution by endonuclease‐assisted target recycling with a Y‐shaped junction DNA structure, thus leading to the correlation of the stripping signal to the target concentration. The functionalized nanosphere was characterized with X‐ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The proposed method showed a linear range from 0.1 to 1000 fM with a limit of detection of 0.066 fM (3σ) and good selectivity for base discrimination. The designed strategy provided a sensitive tool for DNA analysis and could be widely applied in bioanalysis and biomedicine.     

8-Oxoguanosine switches modulate the activity of alkylated siRNAs by controlling steric effects in the major versus minor grooves

Small interfering double-stranded RNAs have been synthesized bearing one or more base modifications at nucleotide positions 4, 11, and/or 16 in the guide strand. The chemically modified base is an N(2)-alkyl-8-oxo-7,8-dihydroguanine (alkyl = propyl, benzyl) that can alternatively pair in a Watson-Crick sense opposite cytosine (C) or as a Hoogsteen pair opposite adenine (A). Cellular delivery with C opposite led to effective targeting of A-containing but not C-containing mRNA sequences in a dual luciferase assay with RNA interference levels that were generally as good as or better than unmodified sequences. The higher activity is ascribed to an inhibitory effect of the alkyl group projecting into the minor groove of double-stranded RNA preventing off-target binding to proteins such as PKR (RNA-activated protein kinase).     

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.     

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.     

Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands

To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.     

Formation and stability of a Janus-Wedge type of DNA triplex

A new type of DNA targeting with the formation of a Janus-Wedge (J-W) triple helix is described. The "wedge" residue (W) attached to a PNA backbone is designed to insert itself into double-stranded DNA and base pair with both Watson-Crick faces. To study the stability of such an assembly, we have examined the formation of the J-W triplex with dC8 - T8 target sequence. The use of this target sequence permits the study of this new helix form without competing Watson-Crick interactions between the two target residues. Studies indicate that the W strand binds to both target strands, with defined polarity and a stability (-15.2 kcal/mol) that is roughly the sum of the two independent duplex interactions.     

Dual-labeled oligonucleotide probe for sensing adenosine via FRET: a novel alternative to SNPs genotyping

A novel FRET based strategy for DNA sequence analysis utilising base-discriminating fluorescence (BDF) nucleoside, (Py)U/(2-Ant)U, as donor in the dual-labelled oligonucleotide probe is reported; a selective/specific emission from acceptor, was observed upon excitation at the donor, only when the opposite base of the "smart" fluorescently labeled BDF nucleoside, (Py)U/(2-Ant)U, is adenine on the complementary target sequence.     

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.     

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.     

Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.     

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.     

Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.

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Graphical abstract Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.

     

DNA logic gates

A conceptually new logic gate based on DNA has been devised. Methoxybenzodeazaadenine ((MD)A), an artificial nucleobase which we recently developed for efficient hole transport through DNA, formed stable base pairs with T and C. However, a reasonable hole-transport efficiency was observed in the reaction for the duplex containing an (MD)A/T base pair, whereas the hole transport was strongly suppressed in the reaction using a duplex where the base opposite (MD)A was replaced by C. The influence of complementary pyrimidines on the efficiency of hole transport through (MD)A was quite contrary to the selectivity observed for hole transport through G. The orthogonality of the modulation of these hole-transport properties by complementary pyrimidine bases is promising for the design of a new molecular logic gate. The logic gate system was executed by hole transport through short DNA duplexes, which consisted of the "logic gate strand", containing hole-transporting nucleobases, and the "input strand", containing pyrimidines which modulate the hole-transport efficiency of logic bases. A logic gate strand containing multiple (MD)A bases in series provided the basis for a sharp AND logic action. On the other hand, for OR logic and combinational logic, conversion of Boolean expressions to standard sum-of-product (SOP) expressions was indispensable. Three logic gate strands were designed for OR logic according to each product term in the standard SOP expression of OR logic. The hole-transport efficiency observed for the mixed sample of logic gate strands exhibited an OR logic behavior. This approach is generally applicable to the design of other complicated combinational logic circuits such as the full-adder.     

A Peptide Nucleic Acid (PNA)‐DNA Ferrocenyl Intercalator for Electrochemical Sensing

A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA‐DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA‐DNA hybrid. The ferrocene oxidation peaks were utilised for the target DNA quantification. The developed biosensor demonstrated a good linear dependence of FND oxidation peak on DNA concentration in the range 1 fM to 100 nM of target DNA, with a low detection limit of 11.68 fM. Selectivity tests were also investigated with a non‐complementary DNA sequence, indicating that the FND intercalator exhibits a selective response to the target PNA‐DNA duplex.     

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.     

Isostable DNA

The high fidelity detection of multiple DNA sequences in multiplex assays calls for duplexes whose stability is independent of sequence (isostable DNA), forming under universally stringent conditions. Nature did not evolve DNA to form isostable duplexes. Here we report how probe strands can be modified so that an all-A/T target strand is bound with the same or slightly higher affinity than the corresponding all-G/C strand with the same sequence of purines and pyrimidines. We refer to these probes that feature covalently attached ligands as "decorated nucleic acids". Caps, intercalators, and locks were used to stabilize A/T duplexes, and N4-ethylcytosine residues were employed to tune down the stability of G/C duplexes without significantly affecting base pairing selectivity. Near-isostability was demonstrated in solution and on microarrays of high and low density. Further, it is shown that hybridization results involving decorated probes on microarrays can be predicted on the basis of thermodynamic data for duplex formation in solution. Predictable formation of isostable DNA not only benefits microarrays for gene expression analysis and genotyping, but may also improve the sequence-specificity of other applications that rely on the massively parallel formation of Watson-Crick duplexes.     

An isothermal strand displacement amplification strategy for nucleic acids using junction forming probes and colorimetric detection

The authors describe a method for DNA target recognition and signal amplification that is based on the target-induced formation of a three way junction. The subsequent assembly of two DNA probes releases the inhibitory strand and triggers a downstream strand displacement amplification. This causes the formation of a G-rich single sequence that binds to a hemin monomer with its peroxidase-mimicking properties. The resulting peroxidase (POx) activity is quantified by using H₂O₂ and TMB as the substrate. In the presence of an inhibitor, in contrast, the POx-like activity is strongly reduced. This forms the basis for a highly sensitive DNA assay. It has a 0.8 pM detection limit when operated at a wavelength of 450 nm and was applied to the isothermal determination of target DNA with high selectivity.

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Graphical abstract Schematic of the assay: Introduction of target results in the formation of a three way junction. The subsequent assembly of two probes releases the inhibitory strand and triggers a downstream strand displacement amplification, generating amount of G-rich single sequence which causes peroxidase-mimicking activity on binding to a hemin monomer.

     

Ultrasensitive Electrochemiluminescence Biosensor for mRNA Based on Polymerase Assisted Signal Amplification

A novel biosensor for ultra‐trace mRNA sensing was constructed based on isothermal circular strand‐replacement polymerization (CSRP) to amplify the electrochmeiluminescence (ECL) signal by combining quantum dots (CdTe) as luminophore. After the hairpin‐like capture DNA was opened by hybridization with target mRNA, the additive primer (DNA1) was able to get access to its complementary sequence which is partially belong to the stem part and triggered a polymerization of DNA strand, leading to the release of target mRNA and another polymerization cycle. The remaining sequence of the stem part continued to hybridize with QDs labeled DNA, accomplishing ECL signal amplification. Target mRNA could be specifically assayed with a linear relationship between the signal intensity and the logarithm of concentrations of target DNA in the range of 1.0×10⁻¹⁴∼5.0×10⁻¹⁰ M, with a low detection limit of 1.4×10⁻¹⁵ M. The signal could discriminate perfect matched target mRNA from 1‐base mismatch sequence. This proposed ECL biosensor exhibited an efficient performance in serum sample, opening new opportunities for genetic target analysis in diagnostic and clinic biomedical fields.