Synthesis and Photophysical Characterisation of a Fluorescent Nucleoside Analogue that Signals the Presence of an Abasic Site in RNA

The synthesis and site‐specific incorporation of an environment‐sensitive fluorescent nucleoside analogue ( 2 ), based on a 5‐(benzofuran‐2‐yl)pyrimidine core, into DNA oligonucleotides (ONs), and its photophysical properties within these ONs are described. Interestingly and unlike 2‐aminopurine (a widely used nucleoside analogue probe), when incorporated into an ON and hybridised with a complementary ON, the emissive nucleoside 2 displays significantly higher emission intensity than the free nucleoside. Furthermore, photophysical characterisation shows that the fluorescence properties of the nucleoside analogue within ONs are significantly influenced by flanking bases, especially by guanosine. By utilising the responsiveness of the nucleoside to changes in base environment, a DNA ON reporter labelled with the emissive nucleoside 2 was constructed; this signalled the presence of an abasic site in a model depurinated sarcin/ricin RNA motif of a eukaryotic 28S rRNA.     

Tropolone-Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism,Enzymatic Incorporation into DNA and HeLa Cell Internalization

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.     

Synthesis,Photophysical Properties and Incorporation of a Highly Emissive and Environment‐Sensitive Uridine Analogue Based on the Lucifer Chromophore

The majority of fluorescent nucleoside analogues used in nucleic acid studies have excitation maxima in the UV region and show very low fluorescence within oligonucleotides (ONs); hence, they cannot be utilised with certain fluorescence methods and for cell‐based analysis. Here, we describe the synthesis, photophysical properties and incorporation of a highly emissive and environment‐sensitive uridine analogue, derived by attaching a Lucifer chromophore (1,8‐naphthalimide core) at the 5‐position of uracil. The emissive nucleoside displays excitation and emission maxima in the visible region and exhibits high quantum yield. Importantly, when incorporated into ON duplexes it retains appreciable fluorescence efficiency and is sensitive to the neighbouring base environment. Notably, the nucleoside signals the presence of purine repeats in ON duplexes with an enhancement in fluorescence intensity, a property rarely displayed by other nucleoside analogues.     

Responsive Fluorescent PNA Analogue as a Tool for Detecting G‐quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome‐Inactivating Proteins

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic‐acid‐based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment‐sensitive 5‐(benzofuran‐2‐yl)‐ and 5‐(benzothiophen‐2‐yl)‐uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene‐modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn‐on detection of G‐quadruplex‐forming promoter DNA sequences of human proto‐oncogenes (c‐myc and c‐kit). Furthermore, the ability of benzothiophene‐modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome‐inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.     

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes

The development of biophysical systems that enable an understanding of the structure and ligand‐binding properties of G‐quadruplex (GQ)‐forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ‐directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H‐Telo) DNA and RNA repeats in a cell‐like confined environment by using conformation‐sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2‐ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2′‐deoxy and ribonucleoside probes, composed of a 5‐benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H‐Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H‐Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy‐to‐handle RMs could provide new opportunities to study and devise screening‐compatible assays in a cell‐like environment to discover GQ binders of clinical potential.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Molecular Design of an Environmentally Sensitive Fluorescent Nucleoside, 3‐Deaza‐2′‐Deoxyadenosine Derivative: Distinguishing Thymine by Probing the DNA Minor Groove

An environmentally sensitive fluorescent nucleoside containing a 3‐deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3‐naphthylethynylated 3‐deaza‐2′‐deoxyadenosine (^3nzA, 1 ) exhibited dual fluorescence emission from an intramolecular charge‐transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.     

Ultramild Protein‐Mediated Click Chemistry Creates Efficient Oligonucleotide Probes for Targeting and Detecting Nucleic Acids

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)‐binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine‐based fluorescent probes in vitro and rationalize our results by electronic structure calculations.     

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification‐free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra‐bright macrofluorophores of 9–84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target‐specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.     

Fluorescent Probes for HOCl Imaging

Small-molecule fluorescent probes for hypochlorous acid (HOCl), one of the poorly understood reactive oxygen species (ROS), help to unveil HOCl functions in health and disease. Numerous small-molecule HOCl fluorescent probes have been developed in the past decade. Nevertheless, only a portion of them demonstrated their practical applications in biomedical research because of common problems in selectivity, sensitivity, chemostability, and photostability, etc. The problems could be addressed by a combination of rational probe design and careful selection of fluorophore templates. In this review, we describe several classes of representative HOCl fluorescent probes based on their fluorophore templates, and we discuss their design strategies, photophysical properties, and biological applications. A comprehension of their strengths, weaknesses, and common uses will facilitate the development of ideal HOCl assays and the discovery of novel biological processes.     

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.     

1,8‐Naphthalimide Derivative Dyes with Large Stokes Shifts for Targeting Live‐Cell Mitochondria

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8‐naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8‐naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt‐4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt‐4 stood out as a potential mitochondrial dye for living‐cell experiments involving induced mitochondrial stress arising from the treatments because Mt‐4 shows enhanced fluorescence in mitochondrial stress situations.     

Stacked Thiazole Orange Dyes in DNA Capable of Switching Emissive Behavior in Response to Structural Transitions

Functional nucleic acids with the capability of generating fluorescence in response to hybridization events, microenvironment or structural changes are valuable as structural probes and chemical sensors. We now demonstrate the enzyme-assisted preparation of nucleic acids possessing multiple thiazole orange (TO) dyes and their fluorescent behavior, that show a spectral change from the typical monomer emission to the excimer-type red-shifted emission. We found that the fluorescent response and emission wavelength of the TO dyes were dependent on both the state of the DNA structure (single- or double-stranded DNA) and the arrangement of the TO dyes. We showed that the fluorescent behavior of the TO dyes can be applied for the detection of RNA molecules, suggesting that our approach for preparing the fluorescent nucleic acids functionalized with multiple TO dyes could be useful to design a fluorescence bioimaging and detection technique of biomolecules.     

A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore ( 1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.     

Two-photon fluorescent probes for biomembrane imaging: effect of chain length

Two-photon fluorescent probes for the cellular membrane, derived from 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), and stearyl (CS) groups as the receptor, have been synthesized. Their photophysical properties and utility as membrane probes were also studied. Whereas CH cannot be used as a membrane probe due to its high water solubility, CL and CS are useful two-photon probes for membrane lateral heterogeneity, as they can easily stain cells, emit fluorescence with high sensitivity to the environment polarity, and are capable of imaging the membrane lateral heterogeneity in live cells. Moreover, CS is more likely to be located in the plasma membrane due to its negligible water solubility. Our results show that the liquid ordered-like domain covers 31-35% of the cellular surface.     

Highly Efficient Quencher‐Free Molecular Beacon Systems Containing 2‐Ethynyldibenzofuran‐ and 2‐Ethynyldibenzothiophene‐Labeled 2′‐Deoxyuridine Units

We have prepared two fluorescent DNA probes—U^DBF and U^DBT, containing 2‐ethynyldibenzofuran and 2‐ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU—and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher‐free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence “turn‐off” response to mismatched targets with guanine bases; this suggests that these probes could be used as base‐discriminating fluorescent nucleotides. On the other hand, we observed a “turn‐on” response to matched targets when the U^DBF and U^DBT units of ODNs containing pyrimidine‐based FBs were positioned opposite the four natural nucleobases. In particular, an ODN incorporating U^DBT and cytosine FBs has potential use in single‐nucleotide polymorphism typing.     

生物标示用3H-吲哚菁染料

随着生物技术和荧光标示技术的飞速发展,3H-吲哚菁染料已成为在DNA、蛋白质及核酸等分析检测中使用的主要荧光探针。其中代表性化合物Cy3.、Cy5.作为新一代商品化荧光标示剂在生物芯片、分子信标等方面得到了重要应用。本文综述了该类碳菁染料在生物领域中取得的重大应用进展、并探讨了其结构与性能之间的关系。     

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis,Hybridization Studies,Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5‐(pyren‐1‐yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene‐modified nucleobase component were found to destabilize an i‐motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene‐modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene‐modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady‐state fluorescence emission studies of oligonucleotides containing pyrene‐modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene‐modified UNA monomers was observed after formation of i‐motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene‐modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.     

Nanoparticulate Photoluminescent Probes for Bioimaging: Small Molecules and Polymers

Recent interest in research on photoluminescent molecules due to their unique properties has played an important role in advancing the bioimaging field. In particular, small molecules and organic dots as probes have great potential for the achievement of bioimaging because of their desirable properties. In this review, we provide an introduction of probes consisting of fluorescent small molecules and polymers that emit light across the ultraviolet and near-infrared wavelength ranges, along with a brief summary of the most recent techniques for bioimaging. Since photoluminescence probes emitting light in different ranges have different goals and targets, their respective strategies also differ. Diverse and novel strategies using photoluminescence probes against targets have gradually been introduced in the related literature. Among recent papers (published within the last 5 years) on the topic, we here concentrate on the photophysical properties and strategies for the design of molecular probes, with key examples of in vivo photoluminescence research for practical applications. More in-depth studies on these probes will provide key insights into how to control the molecular structure and size/shape of organic probes for expanded bioimaging research and applications.