核酸荧光探针TOTO单体噻唑橙的合成研究

研究设计了以 2 巯基苯并噻唑和 4 甲基喹啉为主要原料 ,合成核酸荧光探针TOTO单体噻唑橙 (TO)的工艺路线。采用碘甲烷和对甲苯磺酸甲酯 ,先后对 2 巯基苯并噻唑进行了氮甲基化和巯甲基化反应 ,收率分别为 92 4 %和 91%。其后 ,用 4 甲基喹啉与 1,3 二溴丙烷反应 ,并将其产物与两步甲基化后产物反应 ,合成了目的产物TO ,后两步收率分别为 5 2 5 %和6 1 2 %。经核磁共振谱鉴定产品结构正确 ,从而证明了通过该路线可以合成TOTO单体     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.     

Triplex formation by pyrene-labelled probes for nucleic acid detection in fluorescence assays

Triplex-forming homopyrimidine oligonucleotides containing insertions of a 2'-5' uridine linkage featuring a pyrene moiety at the 3'-position exhibit strong fluorescence enhancement upon binding to double-stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 degrees C and in an increase in the fluorescence quantum yield (Phi(F)) from 0.061 to 0.179, while the probe alone was fluorescently silent (Phi(F)=0.001-0.004). To achieve higher triplex stability, five nucleobases in a 14-mer sequence were substituted with alpha-L-LNA monomers, which provided a triplex with a T(m) of 49.5 degrees C and a Phi(F) of 0.158 at pH 6.0. Under similar conditions, a Watson-Crick-type duplex formed with the latter probe showed lower fluorescence intensity (Phi(F)=0.081) than for the triplex.     

Fluorescent analysis of translesion DNA synthesis by using a novel, non-natural nucleotide analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2-APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real-time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function.     

Real-time detection of nucleotide incorporation during complementary DNA strand synthesis

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.     

Tropolone-Conjugated DNA: Fluorescence Enhancement in the Duplex

Tropolone (2-hydroxycyclohepta-2,4,6-triene-1-one and tautomer) is a non-benzenoid bioactive natural chromophore with pH-dependent fluorescence character and extraordinary metal binding affinities, especially with transition-metal ions Cu²⁺/Zn²⁺/Ni²⁺. This report describes the syntheses and biophysical studies of a new tropolonyl thymidine [(4(5)-hydroxy-5(4)-oxo-5(4)H-cyclohepta-1,3,6-trienyl)thymidine] (tr-T) nucleoside and of corresponding tropolone-conjugated DNA oligonucleotides that form B-form DNA duplex structures with a complementary DNA strand, although their duplex structures are less stable than that of the control. Furthermore, the stabilities of those DNA duplex structures are lowered by the presence of increasing numbers of tr-T residue or by decreasing pH of their environments. Most importantly, these duplex structures are made fluorescent because of the presence of the tropolone moieties conjugated to the thymidine residues. The fluorescence behavior of those duplex structures exhibits pH dependence, with stronger fluorescence at lower pH and weaker fluorescence at high pH. Importantly, the fluorescence characters of tr-DNA oligonucleotides are significantly enhanced by nearly threefold after duplex structure formation with their complementary control DNA oligonucleotide. Further, the fluorescence behavior of these tr-DNA duplex structures is also dependent on the pH conditions. Hence, tropolonyl-conjugated DNA represents a class of new fluorescent analogues that might be be employed for sensing DNA duplex formation and provide opportunities to improve fluorescence properties further.     

Progress on the Physiological Function of Mitochondrial DNA and Its Specific Detection and Therapy

Mitochondrial DNA (mtDNA) is the genetic information of mitochondrion, and its structure is circular double-stranded. Despite the diminutive size of the mitochondrial genome, mtDNA mutations are an important cause of mitochondrial diseases which are characterized by defects in oxidative phosphorylation (OXPHOS). Mitochondrial diseases are involved in multiple systems, particularly in the organs that are highly dependent on aerobic metabolism. The diagnosis of mitochondrial disease is more complicated since mtDNA mutations can cause various clinical symptoms. To realize more accurate diagnosis and treatment of mitochondrial diseases, the detection of mtDNA and the design of drugs acting on it are extremely important. Over the past few years, many probes and therapeutic drugs targeting mtDNA have been developed, making significant contributions to fundamental research including elucidation of the mechanisms of mitochondrial diseases at the genetic level. In this review, we summarize the structure, function, and detection approaches for mtDNA. The most current topics in this field, such as mechanistic exploration and treatment of mtDNA mutation-related disorders, are also reviewed. Specific attention is given to discussing the design and development of these probes and drugs for mtDNA. We hope that this review will provide readers with a comprehensive understanding of the importance of mtDNA, and promote the development of effective molecules for theragnosis of mtDNA mutation-related diseases.     

In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.     

Fluorescence properties of 8-(2-pyridyl)guanine "2PyG" as compared to 2-aminopurine in DNA

Because of their environment-sensitive fluorescence quantum yields, base analogues such as 2-aminopurine (2AP), 6-methylisoxanthopterin (6-MI), and 3-methylisoxanthopterin (3-MI) are widely used in nucleic-acid folding and catalysis assays. Emissions from these guanine mimics are quenched by base-stacking interactions and collisions with purine residues. Fluorescent base analogues that remain highly emissive in folded nucleic acids can provide sensitive means to differentiate DNA/RNA structures by participating in energy transfer from proximal ensembles of unmodified nucleobases. The development of new, highly emissive guanine mimics capable of proper base stacking and base-pairing interactions is an important prerequisite to this approach. Here we report a comparison of the most commonly used probe, 2-aminopurine (2AP), to 8-(2-pyridyl)-2'-deoxyguanosine (2PyG). The photophysical properties of these purine derivatives are very different. 2PyG exhibits enhanced fluorescence quantum yields upon its incorporation into folded nucleic acids--approximately 50-fold brighter fluorescence intensity than 2AP in the context of duplex DNA. Due to its bright fluorescence and compatibility with proper DNA folding, 2PyG can be used to accurately quantify energy-transfer efficiencies, whereas 2AP is much less sensitive to structure-specific trends in energy transfer. When using nucleoside monomers, Stern-Volmer plots of 2AP fluorescence revealed upward curvature of F(0) /F upon titration of guanosine monophoshate (GMP), whereas 2PyG exhibited unusual downward curvature of F(0) /F that resulted in a recovery of fluorescence at high GMP concentrations. These results are consistent with the trends observed for 2PyG- and 2AP-containing oligonucleotides, and furthermore suggest that solutions containing high concentrations of GMP can, in some ways, mimic the high local nucleobase densities of folded nucleic acids.     

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G‐Quadruplexes

We have developed fluorescent protein probes specific for parallel G‐quadruplexes by attaching cyan fluorescent protein to the G‐quadruplex‐binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G‐quadruplexes was characterized. The selective recognition and discrimination of G‐quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.     

Templated Chemistry for Sequence‐Specific Fluorogenic Detection of Duplex DNA

We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple‐helix formation at adjacent positions on a specific purine‐rich target sequence of duplex DNA. One fluorescein‐labeled probe contains an α‐azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double‐stranded DNA, at least for homopurine–homopyrimidine target sites, under native and nondenaturing conditions.     

Polyamide Fluorescent Probes for Visualization of Repeated DNA Sequences in Living Cells

DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole‐imidazole polyamides that bind specifically to the minor groove of double‐stranded DNA (dsDNA) represent an attractive approach for in‐cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel‐shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.     

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites—that is, AATT double strand—in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.     

Modulation of Mitochondriotropic Properties of Cyanine Dyes by in Organello Copper‐Free Click Reaction

Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper‐free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3–Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.     

An Azide‐Modified Nucleoside for Metabolic Labeling of DNA

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and strain‐promoted azide–alkyne cycloaddition (SPAAC) “click” reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5‐azido‐2′‐deoxyuridine (AdU) exhibits a 4 h half‐life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.