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.     

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.     

Triplex Crosslinking through Furan Oxidation Requires Perturbation of the Structured Triple‐Helix

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan‐based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan‐oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan‐containing probes for the selective detection of single‐stranded regions within nucleic acids containing a variety of structural motifs.     

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules,Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

The majority of studies on DNA triple helices have been focused on pH‐sensitive parallel triplexes with Hoogsteen CT‐containing third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA‐containing antiparallel triplex‐forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G‐quadruplexes. In this study, G‐rich oligonucleotides containing 2–4 insertions of twisted intercalating nucleic acid (TINA) monomers are demonstrated to disrupt the formation of G‐quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA‐incorporated oligonucleotides was optimized, the rules of their design were established and the optimal triplex‐forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV‐1 genome; dissociation constants as low as 160 nM are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G‐quadruplex with T_m exceeding 90 °C in the presence of 50 mM NaCl. Here we present a set of rules that help to reach the full potential of TINA‐TFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA.     

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

Chemical probes are small‐molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer‐associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug‐resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein–probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein–probe binding, and covalent protein–probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure–activity relationships to support the development of optimum chemical probes.     

Fluorescent Nucleoside Analogs: Probes for Investigating Nucleic Acid Structure and Function

Base-modified fluorescent nucleoside analog probes have been very valuable in the study of nucleic acid structure and function. Many of them structurally resemble natural bases, and also display useful properties, such as large Stokes shifts and sensitivity to microenvironment changes. Therefore, unlike traditional fluorescence probes, which mostly report global changes, nucleoside analogs, when incorporated into oligonucleotides, can photophysically report changes that occur around the site of interest, at the nucleotide level. In this review, we provide an overview of various strategies that have been employed to design base-modified fluorescent nucleoside analogs. Then we review recent developments and applications of new generation fluorescent nucleoside analogs with a particular focus on the synthesis, photophysical characterizations and applications of heterobicycle-conjugated pyrimidine nucleoside analogs that have been developed by our group. These analogs, which have a minimal effect on the structures of the oligonucleotides into which they are incorporated, show emission in the visible region and excellent fluorescence solvatochromism. Notably, unlike the majority of fluorescent nucleoside analogs developed so far, these analogs retain their fluorescence efficiency when incorporated into oligonucleotides. We anticipate that these nucleoside analogs, with such photophysical properties, would be useful in designing robust biophysical assays to study nucleic acids.     

Tuning the pH Response of i‐Motif DNA Oligonucleotides

Cytosine‐rich single‐stranded DNA oligonucleotides are able to adopt an i‐motif conformation, a four‐stranded structure, near a pH of 6. This unique pH‐dependent conformational switch is reversible and hence can be controlled by changing the pH. Here, we show that the pH response range of the human telomeric i‐motif can be shifted towards more basic pH values by introducing 5‐methylcytidines (5‐MeC) and towards more acidic pH values by introducing 5‐bromocytidines (5‐BrC). No thermal destabilisation was observed in these chemically modified i‐motif sequences. The time required to attain the new conformation in response to sudden pH changes was slow for all investigated sequences but was found to be ten times faster in the 5‐BrC derivative of the i‐motif.     

Stabilization of Telomeric I‐Motif Structures by (2′S)‐2′‐Deoxy‐2′‐C‐Methylcytidine Residues

G‐quadruplexes and i‐motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH‐sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C‐rich oligonucleotides carrying conformationally restricted (2′S)‐2′‐deoxy‐2′‐C‐methyl‐cytidine units. The effect of this modified nucleoside on the stability of intramolecular i‐motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C‐core with C‐modified residues induced the formation of stable intercalated tetraplexes at near‐neutral pH. This study demonstrates the possibility of enhancing the stability of the i‐motif by chemical modification.     

Proteins as Possible Targets for Cytotoxic trans‐Platinum(II) Complexes with Aliphatic Amine Ligands: Further Exceptions to the DNA Paradigm

The reactivity of three cytotoxic trans‐Pt^II complexes bearing aliphatic amine ligands, with transferrin and single‐stranded oligonucleotides as DNA models, was investigated by ESI‐MS and the results obtained are discussed in comparison with cisplatin. Tandem MS studies provided additional information on the preferential Pt binding sites. To determine whether trans‐Pt^II complexes can migrate from a peptide to an oligonucleotide, transfer experiments were also performed using ESI‐MS, and competitive binding of the trans‐Pt^II complexes toward a model peptide and different oligonucleotides was also investigated. Significant differences in the reactivity of the trans complexes with respect to cisplatin were observed. In general, adduct formation with the selected peptide is favored for the trans compounds, whereas cisplatin shows a preference for oligonucleotides, especially if adjacent G–G residues are present. The results are discussed in relation to the possible mechanism of action of the trans‐Pt^II complexes.     

Chemical Probes to Directly Profile Palmitoleoylation of Proteins

Palmitoleoylation is a unique fatty acylation of proteins in which a monounsaturated fatty acid, palmitoleic acid (C16:1), is covalently attached to a protein. Wnt proteins are known to be palmitoleoylated by cis‐Δ9 palmitoleate at conserved serine residues. O‐palmitoleoylation plays a critical role in regulating Wnt secretion, binding to the receptors, and in the dynamics of Wnt signaling. Therefore, protein palmitoleoylation is important in tissue homeostasis and tumorigenesis. Chemical probes based on saturated fatty acids, such as ω‐alkynyl palmitic acid (Alk‐14 or Alk‐C₁₆), have been used to study Wnt palmitoleoylation. However, such probes require prior conversion to the unsaturated fatty acid by stearoyl‐CoA desaturase (SCD) in cells, significantly decreasing their selectivity and efficiency for studying protein palmitoleoylation. We synthesized and characterized ω‐alkynyl cis‐ and trans‐palmitoleic acids (cis‐ and trans‐Alk‐14:1) as chemical probes to directly study protein palmitoleoylation. We found that cis‐Alk‐14:1 could more efficiently label Wnt proteins in cells. Interestingly, the DHHC family of palmitoyl acyltransferases can charge both saturated and unsaturated fatty acids, potentially using both as acyl donors in protein palmitoylation and palmitoleoylation. Furthermore, proteomic analysis of targets labeled by these probes revealed new cis‐ and trans‐palmitoleoylated proteins. Our studies provided new chemical tools and revealed new insights into palmitoleoylation in cell signaling.     

Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets

We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization of the probes and excellent fluorescence responses to single-base mismatches in DNA/RNA targets are demonstrated in model dual-probe and doubly labeled probe formats. This stimulated us to develop two diagnostic systems for the homogeneous detection of a drug-resistance-causing mutation in HIV-1 protease cDNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges of the natural targets, that is, the presence of additional mutations, neighboring sequence variation, and low target concentration, which typically reduce binding and effectiveness of sensing by fluorescent oligonucleotides.     

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.     

Comparison of the Reactivity of Carbohydrate Photoaffinity Probes with Different Photoreactive Groups

A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule–protein interactions. A set of carbohydrate‐based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand‐dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a low‐affinity binding protein in cell lysate.     

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.     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.     

Triplexes with 8‐Aza‐2′‐Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

The fluorescent 8‐aza‐2′‐deoxyisoguanosine ( 4 ) as well as the parent 2′‐deoxyisoguanosine ( 1 ) were used as protonated dCH⁺ surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH⁺ are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4 . Third‐strand binding of oligonucleotides with 4 , opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third‐strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third‐strand binding more flexibility.     

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.     

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.     

DNA‐Directed Assembly of Antibody–Fluorophore Conjugates for Quantitative Multiparametric Flow Cytometry

Multiparametric flow cytometry offers a powerful approach to single‐cell analysis with broad applications in research and diagnostics. Despite advances in instrumentation, progress in methodology has lagged. Currently there is no simple and efficient method for antibody labeling or quantifying the number of antibodies bound per cell. Herein, we describe a DNA‐directed assembly approach to fluorescent labeling that overcomes these barriers. Oligonucleotide‐tagged antibodies and microparticles can be annealed to complementary oligonucleotides bearing fluorophores to create assay‐specific labeling probes and controls, respectively. The ratio of the fluorescence intensity of labeled cells to the control particles allows direct conversion of qualitative data to quantitative units of antibody binding per cell. Importantly, a single antibody can be labeled with any fluorophore by using a simple mix‐and‐match labeling strategy. Thus, any antibody can provide a quantitative probe in any fluorescent channel, thus overcoming major barriers to the use of flow cytometry as a technique for systems biology and clinical diagnostics.