Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT₂₀ as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature (T_m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T_m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.     

Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy

The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.Dedicated to the memory of Wilhelm Fresenius.     

基于锁核酸的高选择性新型分子信标

通过在分子信标的错配位点修饰锁核酸, 不仅可有效地改善其单碱基错配识别能力, 还可提高检测灵敏度. 因而有望发展成为一种通用的提高分子信标单碱基错配识别能力的方法.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

Improved DNA Clamps by Stacking to Adjacent Nucleobases

Three or four aromatic rings interconnected by acetylene bridges form a stiff conjugated system with sufficient conformational freedom to make it useful to link together the two strands of a DNA clamp. Upon targeting a ssDNA, the conformational flexibility allows better stacking of the linker to the underlying non‐planar base triplet in the formed triplex. This type of triplexes has a substantially higher thermal melting temperature which can be further improved by inserting locked nucleic acids (LNAs) in the Hoogsteen part of the clamp. An extremely high sensitivity to mismatches is observed in an octamer triplex when placed in the middle of the sequence.     

Investigation of surface oligonucleotide-binding proteins of eucaryotic cells by affinity modification with reactive oligonucleotide derivatives

Interactions between oligodeoxyribonucleotides (ODN) with different sequences and cell proteins were examined using the affinity modification by [³²P]-labeled reactive oligonucleotide derivatives. 3"-Terminal ribouridine oxidized with sodium periodate, 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine, and the maleimide residue were used as reactive groups. All the compounds used are specific reagents. The set of the discovered nucleic acid-binding (NA-binding) proteins depends on the chemical properties of the affinity reagent. The presence of the hydrophobic group at the 5"-terminus of the ODN molecule is the key factor determining the variety of the discovered NA-binding proteins. The cells of different origin (A431, HeLa, KB, MCF-7, Hap-2, K562, Cos-7, NIH/3T3, human-lung primary epithelial cells, and porcine kidney primary cells) are characterized by the same set of NA-binding proteins whose affinity modifications depends on the conditions of incubation of oligonucleotides with the cells. Treatments of cells disturbing the integrity of the cellular membrane (scrapping, treatment with trypsin, or cell permeabilization with streptolysin O or saponin), disrupt interactions between NA-binding proteins from native cells and ODN.     

DNA电化学传感器在DNA损伤研究中的应用

利用单链DNA分子共价固定在石墨电极表面,采用核酸分子杂交技术,以道诺霉素作为杂交指示剂形成DNA电化学传感器.研究了在不同致突变剂的作用下,特定碱基序列的DNA在电极表面能否杂交及杂交程度的差异,探讨了DNA突变情况及可能的突变机理.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

Universal fluorescent labeling of amplification products using locked nucleic acids

Amplification/hybridization‐based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two‐step amplification process that uses locus‐specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(‐47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(‐21) sequences at lower Tm values). Moreover, AT‐rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC‐rich substitutions. GC‐rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA‐containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.     

Allosteric control of oligonucleotide hybridization by metal-induced cyclization

A bis(terpyridine) modified single-stranded DNA smoothly forms a stable cycle by Fe2+-assisted ring closure. A novel, complex type of allosteric behavior is observed with Zn2+, which reversibly off-regulates binding of a complementary oligonucleotide only in a narrow concentration range.     

A designed locked nucleic acid-based nanopore for discriminating ctDNA and its coexisting analogue ncDNA

Circulating tumor DNA(ctDNA),carrying tumor-specific sequence mutations,is a promising biomarker for classification,diagnosis and prognosis of cancers.However,there is still a great challenge in discriminating single-base difference between ctDNA and its coexisting analogue(normal circulating DNA,ncDNA)at a serum sample.A locked nucleic acid(LNA)probe combined with a-HL nanopore sensor was designed,which achieved a high signal-to-background ratio(SBR)of^8.34 × 10^3,as well as a significant discrimination capability(~12.3 times)of single-base diffe rence.The accurate discrimination strategy is label-free,convenient,selective and sensitive,which has great potential in the early diagnosis of diseases and biomedical research fields.     

A fluorescent molecular switch for room temperature operation based on oligonucleotide hybridization without labeling of probes or targets

A molecular switch was prepared by self-assembly. Neutravidin served as a template that allowed for a biotinylated probe oligonucleotide to be placed adjacent to a biotinylated long-chain linker that was terminated with thiazole orange (TO). Hybridization of probe oligonucleotide with target to form double-stranded DNA resulted in intercalation of the adjacent TO probe. This was a reversible process that could be tracked by fluorescence intensity changes. Formamide was used as a denaturant for double-stranded DNA, and could be used to depress thermal denaturation temperatures. In this work formamide had a dual function, providing for control of hybridization selectivity at room temperature, while concurrently ameliorating non-specific adsorption to improve signal-to-noise when using thiazole orange as a fluorescence signalling agent to determine oligonucleotide hybridization. Room temperature single nucleotide polymorphism (SNP) discrimination for oligonucleotide targets was achieved both in solution and for molecular switches that were immobilized onto optical fibers. In solution, a concentration of 18.5% formamide provided greater than 40-fold signal difference between single-stranded DNA and double-stranded DNA, in contrast to only a 2-fold difference in the absence of formamide. Selectivity for SNP determination in solution was demonstrated using targets of varying lengths including a 141-base PCR amplicon. The improved signal-to-noise achieved by use of formamide is likely due to preferential displacement of dye molecules that are otherwise electrostatically bound to the polyanionic nucleic acid backbone.     

Selective recognition of Ca ions using novel polymeric phenols

The research described here elucidates the fundamental interactions of various cations with phenolic moieties constituting the side chains of novel copolymers. The phenolic group was chosen because similar interactions of the tyrosine (Tyr) moiety in proteins with alkaline earth cations are of particular interest for some biochemical systems where both the alkaline earth cations and the aromatic compounds are abundant. The present study has revealed the preferred binding site for our polymeric systems.     

Selective recognition of CG interruption by 2′,4′-BNA having 1-isoquinolone as a nucleobase in a pyrimidine motif triplex formation

To develop a novel nucleoside analogue for the effective recognition of CG interruption in a homopurine-homopyrimidine tract of double-stranded DNA (dsDNA), we succeeded in the synthesis of a triplex-forming oligonucleotide (TFO) containing a novel 2′,4′-BNA (Q^B) bearing 1-isoquinolone as a nucleobase, and the triplex-forming ability and sequence-selectivity of the TFO (TFO-Q^B) were examined. On melting temperature (T_m) measurements, it was found that the TFO-Q^B formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.     

Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling.     

Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe

This communication reports on a novel biosensor to study the hybridization specificity by using thiolated hairpin locked nucleic acids (LNA) as the capture probe. The LNA probe was immobilized on the gold electrode through sulfur–Au interaction and could selectively hybridize with its target DNA. Differential pulse voltammetry (DPV) was used to monitor the hybridization reaction on the probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This LNA probe has been used for assay of fusion gene in Chronic Myelogenous Leukemia (CML) of the real sample with satisfactory result.     

Magnetic bead-based hybridization assay for electrochemical detection of microRNA

Aberrant expression of microRNAs (miRNAs), short non-coding RNA molecules regulating gene expression, is often found in tumor cells, making the miRNAs suitable candidates as cancer biomarkers. Electrochemistry is an interesting alternative to current standard methods of miRNA detection by offering cheaper instrumentation and faster assays times. In this paper, we labeled miRNA in a quick, simple, two-step procedure with electroactive complex of osmium(VI) and 2,2′-bipyridine, Os(VI)bipy, which specifically binds to the ribose at the 3′-end of the miRNA, and hybridized such labeled miRNA with biotinylated capture probe attached to the streptavidin magnetic beads. Labeled miRNA was then detected at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of the peak from the Os(VI)bipy label. We obtained good selectivity of the assay using elevated hybridization temperatures for better discrimination of perfect duplex from single and double mismatches. After optimization of the protocol, we demonstrated feasibility of our assay by detecting target miRNA in real total RNA samples isolated from human cancer cells.     

Benzoisothiazole-1,1-dioxide-based synthetic receptor for zinc ion recognition in aqueous medium and its interaction with nucleic acids

Benzoisothiazole-1,1-dioxide-based synthetic receptor was prepared by two step synthesis in 92 % overall yield. Its applicability for the determination of Zn(II) and interction with nucleic acids was studied by absorption spectroscopy. Obtained data, specifically low limit of detection, 0.15 µM (R² = 0.9933), showed the high potential of the tested structure motif for the recognition and determination of Zn(II) ions in aqueous media (water:DMSO; 99:1 (ν/ν)). Alone receptor displayed orderly strongly RNA affinity. Value of LogK was 6.1 and 4.9 for its complex (1:1) with RNA and DNA, respectively. Nevertheless, in presence of complexed Zn(II) ions, its DNA affinity (represent by K, LogK = 5.7) strongly grow to near value obtained for its interaction with RNA. On the other hand, its RNA affinity (LogK = 5.9) displayed not significantly change in the presence of complexed one.