Oligodeoxynucleotide Probes Research Articles

Enhanced Sequence-Specific DNA Recognition Using Oligodeoxynucleotide-Benzimidazole Conjugates.

Synthetic modification of oligodeoxynucleotides (ODNs) via conjugation to nucleic acid binding small molecules can improve hybridization and pharmacokinetic properties. In the present study, five Hoechst 33258 derived benzimidazoles were conjugated to T rich ODNs and their hybridization effectiveness was tested. Thermal denaturation studies revealed significant stabilization of complementary duplexes by ODN-benzimidazole conjugates, with the extent of stabilization being highly dependent on the length of the linker between DNA and benzimidazole. The increases in thermal stability were determined to be due to the binding of the benzimidazole moiety to the duplex. Circular dichroism and molecular modeling studies provided insights toward the influence of conjugation on duplex structure and how linker length impacts placement of the benzimidazole moiety in the minor groove. Furthermore, thermal denaturation studies with the complementary strand containing a single base mismatch or being RNA revealed that covalent conjugation of benzimidazoles to an ODN also enhances the sequence specificity. The fundamental studies reported herein provide a strategy to improve the stability and specificity properties of the ODN probes, which can be of use for targeting and diagnostics applications.

Acyclic 2-Ethynylnaphthalene-Modified 8-Aza-3,7-dideaza-2′-deoxyadenosine Allows Thymine Discrimination by Probing the DNA Minor Groove Environment

AbstractTwo novel acyclic environment-sensitive fluorescent nucleosides named ac37zA and an37zA are synthesized and their photophysical properties are investigated. Both ac37zA and an37zA exhibit dual fluorescence emission depending upon molecular coplanarity. Oligodeoxynucleotide probes containing ac37zA clearly discriminate perfectly matched thymine in target DNA strands by strong charge transfer (CT) emission in the longer wavelength region and by strong quenching of emission from the twisted conformation in the mismatched case.

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives.

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.

Consequences of isosteric replacement of benzene for pyrazine in phthalocyanines

Tetrapyrazinoporphyrazines (TPyzPzs) likely represent the most investigated group of azaanalogs of phthalocyanines. This review highlights the author’s contributions in this field and summarizes the most important features of these interesting macrocycles with the aim of emphasizing the differences from the parent phthalocyanines. In particular, uniqueness in synthesis, spectral, photophysical and electrochemical properties are discussed. Thanks to systematic investigation of intramolecular charge transfer with TPyzPzs as electron acceptors, detailed structure-activity relationships of this ultrafast quenching process have been disclosed. This opened the door for the use of TPyzPzs as fluorescence sensors and quenchers in oligodeoxynucleotide probes, the latter being unique for TPyzPzs, which have not been investigated with phthalocyanines. These two applications seem to be much more suitable for TPyzPzs than photodynamic therapy, where the phthalocyanines typically have superior in vitro activities.

Comparison of Quenching Efficiencies in Long Triple-Labeled and Double-Labeled TaqMan Oligodeoxynucleotide Probes.

Although triple labeling of molecular beacons has been documented to improve quenching efficiencies and studies generally assume similar benefits at long TaqMan probes, a limited number of works have studied this issue in TaqMan probes. We therefore prepared a series of long triple-labeled oligodeoxynucleotide probes with 6-carboxyfluorescein as a fluorophore at the 5'-end and BlackBerry (BBQ-650) or azaphthalocyanine quenchers at the 3'-end and in the intrastrand position and systematically compared their quenching efficiencies with those of the corresponding double-labeled probes including important control probes. A model polymerase chain reaction (PCR) assay enabled the determination of the quenching efficiencies of static and Förster resonance energy transfer (FRET) quenching in the target probes. The type of probe had no effect on the static quenching ability. Importantly, FRET quenching of double-labeled probes with a quencher at the 3'-end showed a statistically insignificant difference from the control probe without any quencher, indicating the need to shift the quencher closer to the fluorophore in long probes. Shortening the distance between the fluorophore and the quencher played a key role in FRET quenching, whereas the introduction of an additional quencher only slightly improved the quenching efficiency. BBQ-labeled probes had lower quenching efficiencies than azaphthalocyanine probes. The methodologies and relationships described above seem, however, to be universal and applicable to any quencher.

FRET-based dual-labeled oligonucleotide probes detect target DNA by probing a minor groove environment.

A novel amino-modifier ESF nucleoside AM37zA (1) containing a trifluoroacetyl (TFA)-protected amino group is designed for the functionalization of ODN probes after oligonucleotide synthesis. AM37zA (1) showed remarkable solvatochromicity and ODN probes containing deprotected AM37zA [ODN(N37zA)] discriminated single base alteration in target DNA based on changes in the fluorescence wavelength and intensity. Using AM37zA (1), a FRET-based dual-labeled ODN probe, ODN(F37zA), which contains two fluorophores at the major and minor groove sides of DNA, has been synthesized. The ODN(F37zA) incorporated with fluorescein by post-synthetic modification discriminated the target DNA sequence by a distinct change in the fluorescence intensity (S/N ratio of 20 : 1). Thus, an amino-modifier, AM37zA, and a FRET-based ODN probe, ODN(F37zA), are valuable for monitoring the microenvironment around nucleic acids and can act as powerful tools for examining the microstructures of DNA/RNA, including gene detection.

The chromatographic behaviour of new double-labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity.

The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.

Fluorene‐Labeled 2'‐Deoxyuridine as an Environmentally Sensitive Probe for Detection of an Abasic Site

AbstractThe fluorescence properties of the fluorene‐labeled 2'‐deoxyuridine derivative UF are sensitive to the polarity and viscosity of its solvent. We incorporated this environment‐sensitive unit into the central position of oligodeoxynucleotides (ODNs) and placed various flanking bases (FBs) around the UF residue, then investigated the changes in fluorescence that occurred when these ODN probes encountered DNA targets containing an abasic site. The ODN probe featuring cytosine‐FBs turned on its fluorescence when contacting the fully matched target DNA sequence, but turned off its fluorescence dramatically when the target DNA contained an abasic site.

Synthesis of ethynylpyrene-modified 3-deaza-2′-deoxyguanosines as environmentally sensitive fluorescent nucleosides: Target DNA-sequence detection via changes in the fluorescence wavelength

Novel pyrene-labeled environmentally sensitive fluorescent (ESF) nucleosides [1p3zG (1a) and 2p3zG (1b)] possessing the 3-deazaguanine skeleton were synthesized. Among them, 1p3zG (1a) exhibited longer fluorescence emission wavelengths compared with the previously reported naphthalene-modified ESF nucleoside 3nzG (48–71 nm longer in polar solvents) and showed remarkable solvent-polarity-sensitive fluorescence properties. Oligodeoxynucleotide (ODN) probes containing 1p3zG (1a) clearly discriminated target DNAs by the change in the emission wavelength and intensity through monitoring the microenvironmental change around the DNA minor groove. Thus, these pyrene-labeled ESF nucleosides can be applied in gene detection and molecular diagnostics, as well as in the structural analysis of nucleic acids.

In Situ Hybridization in Mineralized Tissues: The Added Value of LNA Probes for RNA Detection.

In situ hybridization (ISH) is one of the fundamental methods in developmental biology and neurobiology. Their first ISH protocols were reported in 1969 (Gall and Pardue, Proc Natl AcadSci USA 63:378-83, 1969). Since several decades, ISH based on the specific hybridization of 100-2000 nucleotides long probes enabled the localization of DNA/RNA sequences in tissues and cells with high cellular resolution. But sometimes a limited sensitivity notably in mineralized tissues (Obernosterer et al., Nature Protocols 2:1508-14, 2007).Here we describe a recent improvement of in situ hybridization efficiency by applying nucleotide locked nucleic acid (LNA)-incorporated oligodeoxynucleotide probes (20 LNA/DNA nucleotide probes) essentially used for noncoding miRNA and messenger RNAs.

Spectroscopic study of fluorescent probes based on G-quadruplex oligonucleotides labeled with ethynylpyrenyldeoxyuridine

The design, synthesis, and spectral properties of four pyrene labeled oligonucleotide probes with G-quadruplex structure (Tel22–Tpy, Tel22–Upy, Tel22–6Upy, Tel22–18Upy) based on the 22-mer human telomeric sequence (Tel22) have been reported. Pyrene labels in the form of ethynylpyrenyldeoxyuridine have been inserted efficiently into oligodeoxynucleotides probes using phosphoramidite chemistry. The probes exhibited abilities to fold into G-quadruplex structures and to bind metal cations (Na+ and K+). Folding properties of probes and their spectral behavior were examined by recording the UV–vis, fluorescence, and CD spectra as well as by analyzing melting profiles. Fluorescence characteristics and G-quadruplex folding of probes were also studied at the interface of cationic dioctadecyldimethylammonium bromide (DODAB) monolayer. Investigations included film balance measurements (π–A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter.

C2-substituted 8-aza-7-deaza-2′-deoxyadenosines as environmentally sensitive fluorescent nucleosides for discriminating apurinic/apyrimidinic sites in DNA duplex

We synthesized various C2-naphthylethynylated 8-aza-7-deaza-2′-deoxyadenosines 1a–1c as novel environmentally sensitive fluorescent (ESF) purine nucleosides. In particular, the N,N-dimethylamino-substituted derivative 1c exhibits a remarkably high solvatochromicity (Δλ=111nm). 1c-containing oligodeoxynucleotide (ODN) probes clearly discriminated an apurinic/apyrimidinic site (AP site) in the complementary strand via a change in emission wavelength and intensity when hybridized with a target ODN.

Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U(p)) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes successfully distinguished target RNA from single-mutated RNA analyte during an in vitro assay of RNA synthesis.

Hybridization Histochemistry of Neural Transcripts.

Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (or in situ hybridization) permits localization of these transcripts with cellular resolution or better. Furthermore, the relative amounts of transcripts detected in different tissues or in the same tissues in different states (e.g., physiological or developmental) may be quantified. This unit describes hybridization histochemical techniques using either oligodeoxynucleotide probes (see Basic Protocols 1 and 2, Alternate Protocol 1) or RNA probes (riboprobes; see Basic Protocols 3 and 5). These methods include a more recent approach using commercially available sets of oligodeoxynucleotide pairs for colorimetric and fluorescent detection (see Basic Protocol 2), as well as a method for detection of the Y chromosome using either mouse or human riboprobes (see Basic Protocol 5). Additional methods include colorimetric detection (see Basic Protocol 4) and tyramide signal amplification (TSA) of digoxigenin-labeled probes (see Alternate Protocol 2), and autoradiographic detection of radiolabeled probes (see Basic Protocol 6). Finally, methods are provided for labeling oligodeoxynucleotide (see Support Protocol 1) and RNA (see Support Protocol 2) probes, and verifying the probes by northern analysis (see Support Protocol 3).

Design and synthesis of a novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside for monitoring base-pair-induced protonation with cytosine: distinguishing cytosine via changes in the intensity and wavelength of fluorescence.

A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside (BIQ)A (1) comprising a 3-deaza-2'-deoxyadenosine skeleton was developed and used to monitor (BIQ)A-C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized (BIQ)A exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pKa 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of (BIQ)A occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting (BIQ)A-C base pairs were stable. By monitoring the protonation of (BIQ)A under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2'-deoxyadenosine (3z)A forms a stable base pair with cytosine via N(1) protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, (3z)A-containing ODNs could clearly discriminate cytosine through melting temperature (Tm) measurements. Therefore, ODN probes containing indicator nucleosides, such as (BIQ)A and (3z)A, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.

Sensitive Detection of Single-Nucleotide Mutation in the BRAF Mutation Site (V600E) of Human Melanoma Using Phosphate-Pyrene-Labeled DNA Probes.

A series of novel nucleotide phosphoramidites were rationally designed and synthesized and were then site-specifically incorporated in DNA oligonucleotide probes with pyrene-modified phosphate. These oligodeoxynucleotide (ODN) probes almost have no inherent fluorescence emission with pyrene modification at 3' phosphate of corresponding nucleotides as a result of the photoinduced electron-transfer quenching effect of nucleobases (thymidine ∼ cytidine > guanosine ≫ adenosine). However, strong fluorescence emission was observed only with the perfectly matched duplex for the probes with pyrene modified at 3' phosphate of thymidine and cytidine. These rationally designed ODN probes successfully worked as "turn on" fluorescence oligonucleotide sensors for single-nucleotide polymorphism (SNP) and were used for detecting a single BRAF mutation site (V600E) of human melanoma.

Design and synthesis of environmentally sensitive fluorescent 2-naphthylethynylated 2'-deoxyadenosines: Detection of target DNA via changes in fluorescence intensity and wavelength.

Various C2-naphthylethynylated 2'-deoxyadenosines were synthesized as environmentally sensitive fluorescent (ESF) nucleosides and their photophysical properties were examined. Among the ESF nucleosides synthesized, four exhibited strong solvatochromicity, two of which were incorporated into oligodeoxynucleotides (ODNs). These ODN probes were able to detect target DNA through distinct changes in fluorescence intensity and wavelength and acted as effective reporter probes.

Enzyme catalytic amplification of miRNA-155 detection with graphene quantum dot-based electrochemical biosensor.

A specific and sensitive method was developed for quantitative detection of miRNA by integrating horseradish peroxidase (HRP)-assisted catalytic reaction with a simple electrochemical RNA biosensor. The electrochemical biosensor was constructed by a double-stranded DNA structure. The structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture DNA), assembled on the gold electrode surface, with target DNA and aminated indicator probe (NH2-DNA). After the construction of the double-stranded DNA structure, the activated carboxyl groups of graphene quantum dots (GQDs) assembled on NH2-DNA. GQDs were used as a new platform for HRP immobilization through noncovalent assembly. HRP modified biosensor can effectively catalyze the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), accompanied by a change from colorless to blue in solution color and an increased electrochemical current signal. Due to GQDs and enzyme catalysis, the proposed biosensor could sensitively detect miRNA-155 from 1 fM to 100 pM with a detection limit of 0.14 fM. High performance of the biosensor is attributed to the large surface-to-volume ratio, excellent compatibility of GQDs. For these advantages, the proposed method holds great potential for analysis of other interesting tumor makers.

Site-specific modification of the 6-amino group of adenosine in RNA by an interstrand functionality-transfer reaction with an s-functionalized 4-thiothymidine.

Non-natural RNA modifications have been widely used to study the function and structure of RNA. Expanding the study of RNA further requires versatile and efficient tools for site-specific RNA modification. We recently established a new strategy for the site-specific modification of RNA based on a functionality-transfer reaction between an oligodeoxynucleotide (ODN) probe and an RNA substrate. 2'-Deoxy-6-thioguanosine was used to anchor the transfer group, and the 4-amino group of cytosine or the 2-amino group of guanine was specifically modified. In this study, 2'-deoxy-4-thiothymidine was adopted as a new platform to target the 6-amino group of adenosine. The (E)-pyridinyl vinyl keto transfer group was attached to the 4-thioT in the ODN probe, and it was efficiently and specifically transferred to the 6-amino group of the opposing adenosine in RNA in the presence of CuCl2 . This method expands the available RNA target sites for specific modification.

Synthesis and photophysical properties of pyrene-labeled 3-deaza-2'-deoxyadenosines comprising a non-π-conjugated linker: fluorescence quenching-based oligodeoxynucleotide probes for thymine identification.

Pyrene-labeled 3-deaza-2'-deoxyadenosine comprising a non-π-conjugated linker (py3z)A (1) was synthesized and its photophysical properties were investigated. Oligodeoxynucleotide (ODN) probes containing (py3z)A (1) exhibited remarkable fluorescence quenching only when the opposite base of the complementary strand was the perfectly matched thymine. Such fluorescence quenching-based ODN probes exhibited excellent on-off switching properties, making them useful tools for single nucleotide polymorphism (SNP) genotyping and for the identification of target genes and structural studies of nucleic acids.