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.     

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.     

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (^PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without ^PeU modification; thus suggesting that structural distortion is not induced by the modification. If two ^PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three ^PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.     

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.     

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.     

基于生命体系中氨基键合标记的荧光探针研究进展

氨基是生物分子化学结构中常见的基团。通常可利用荧光探针的静电吸引、疏水作用和化学键合的方式对其进行荧光标记。本文综述了基于化学键合作用的荧光探针及其常见的键合类型和染料母体结构。有48篇参考文献。     

Sequence‐Specific Covalent Capture Coupled with High‐Contrast Nanopore Detection of a Disease‐Derived Nucleic Acid Sequence

Hybridization‐based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence‐specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease‐relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap‐containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast—in essence, digital—signal that enables sensitive, single‐molecule sensing of the cross‐linked duplexes.     

Advances in Quantitative FRET‐Based Methods for Studying Nucleic Acids

Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for “on/off” experiments. To use FRET to obtain quantitative distances and three‐dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single‐molecule levels.     

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.     

Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.     

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.     

Fluorescent Probes to Characterise FK506‐Binding Proteins

Talented all‐rounders : Fluorescence polarisation assays were developed for members of the FK506‐binding protein family by using fluorescent rapamycin analogues (demonstrated in the figure). These tracers retain medium to high affinity to all tested proteins (FKBP12, ‐12.6, ‐13, ‐25, ‐51, ‐52). They can be used for active‐site titrations, competition assays with unlabelled ligands and enable a robust, miniaturized assay adequate for high‐throughput screening.

     

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.     

Readily Accessible Fluorescent Probes for Sensitive Biological Imaging of Hydrogen Peroxide

Hydrogen peroxide is a major component of oxygen metabolism in biological systems that, when present in high concentrations, can lead to oxidative stress in cells. Noninvasive molecular imaging of H₂O₂ using fluorogenic systems represents an effective way to detect and measure the accumulation of this metabolite. Herein, we detail the development of robust H₂O₂‐sensitive fluorescent probes using a boronic ester trigger appended to the fluorophore through a benzyl ether linkage. A major advantage of the probes presented here is their synthetic accessibility, with only one step needed to generate the probes on the gram scale. The sensitivity of the probes was evaluated in simulated physiological conditions, showing micromolar sensitivity to H₂O₂. The probes were tested in biological model systems, demonstrating effective imaging of unstimulated, endogenous H₂O₂ levels in RAW 264.7 cells and murine brain tissue.     

Development of Ratiometric Fluorescent Probes for Phosphatases by Using a pKa Switching Mechanism

A novel design strategy for fluorescent probes based on a pK_a switching mechanism was developed. Using this strategy, we developed ratiometric probes for the detection of acid phosphatase activity.

     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

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.