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.     

Quenching of fluorescent nucleobases by neighboring DNA: the "insulator" concept

Fluorescent nucleosides are widely used as probes of biomolecular structure and mechanism in the context of DNA, but they often exhibit low quantum yields because of quenching by neighboring DNA bases. Here we characterize the quenching by DNA of fluorescent nucleosides that have pyrene (Y), perylene (E), benzopyrene (B), or 2-aminopurine (2AP) as nucleobase replacements, and we investigate the effect of inserting varied nucleosides as potential "insulators" between the fluorescent nucleosides and other nearby DNA bases as a strategy for increasing quantum yields. The data show that the hydrocarbons are quenched by adjacent pyrimidines, with thymine being the strongest quencher. The quantum yield of pyrene is quenched 120-fold by a single adjacent T, that of benzopyrene tenfold, and that of perylene by a factor of 2.5. Quenching of excimer and exciplex dinucleoside labels (Y-Y, Y-E, E-E, etc.) was considerably lessened, but was strongest with neighboring thymine. 2-Aminopurine (2AP) is most strongly quenched (15-fold) by neighboring G. We tested four different insulator candidates for reducing this quenching by measuring the fluorescence of short oligonucleotides containing insulators placed between a fluorescent base and a quenching base. The insulators tested were a C(3) abasic spacer (S), dihydrothymidine nucleoside (DHT), terphenyl nucleoside (TP), and adenine deoxynucleoside (dA). Results showed that the abasic spacer had little effect on quenching, while the other three had substantial effects. DHT and terphenyl enhanced fluorescence of the fluorophores by factors of 5 to 70. Adenine base reduced the quenching of pyrene 40-fold. The results underscore the importance of the nearest neighbors in DNA-quenching mechanisms, and establish simple strategies for enhancing fluorescence in labeled DNAs.     

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.     

Forced intercalation probes (FIT Probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection

Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes, in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.     

"Parallel" and "antiparallel tail-clamps" increase the efficiency of triplex formation with structured DNA and RNA targets

Sequence-specific triple-helix structures can be formed by parallel and antiparallel DNA clamps interacting with single-stranded DNA or RNA targets. Single-stranded nucleic acid molecules are known to adopt secondary structures that might interfere with intermolecular interactions. We demonstrate the correlation between a secondary structure involving the target--a stable stem predicted by in silico folding and experimentally confirmed by thermal stability and competition analyses--and an inhibitory effect on triplex formation. We overcame structural impediments by designing a new type of clamp: "tail-clamps". A combination of gel-shift, kinetic analysis, UV thermal melting and thermodynamic techniques was used to demonstrate that tail-clamps efficiently form triple helices with a structured target sequence. The performance of parallel and antiparallel tail-clamps was compared: antiparallel tail-clamps had higher binding efficiencies than parallel tail-clamps both with structured DNA and RNA targets. In addition, the reported triplex-stabilizing property of 8-aminopurine residues was confirmed for tail-clamps. Finally, we discuss the possible use of this improved triplex technology as a new tool for applications in molecular biology.     

2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a K_d of 15.0 mM glucose, which was comparable with the 10 mM K_d previously reported using the strand displacement assay. The binding required two Na⁺ ions or one Mg²⁺ that cannot be replaced by Li⁺ or K⁺. The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K_d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.     

NBD‐Based Green Fluorescent Ligands for Typing of Thymine‐Related SNPs by Using an Abasic Site‐Containing Probe DNA

The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N¹‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH₂) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×10⁶ M ^?1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH₂ is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine).     

2-Methoxypyridine as a Thymidine Mimic in Watson–Crick Base Pairs of DNA and PNA: Synthesis,Thermal Stability,and NMR Structural Studies

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A–T base pair. The hypothesis was that including protonation in the Watson–Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA–DNA duplexes and ≈20 °C in PNA–DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P–A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required.     

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.     

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.     

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.     

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.     

Functionalization of manganite nanoparticles and their interaction with biologically relevant small ligands: picosecond time-resolved FRET studies

We report molecular functionalization of the promising manganite nanoparticles La0.67Sr0.33MnO3 (LSMO) for their solubilization in aqueous environments. The functionalization of individual NPs with the biocompatible citrate ligand, as confirmed by Fourier transform infrared (FTIR) spectroscopy, reveals that citrates are covalently attached to the surface of the NPs. UV-VIS spectroscopic studies on the citrate functionalized NPs reveals an optical band in the visible region. Uniform size selectivity (2.6 nm) of the functionalization process is confirmed from high resolution transmission electron microscope (HRTEM). In the present study we have used the optical band of the functionalized NPs to monitor their interaction with other biologically important ligands. F?rster resonance energy transfer (FRET) of a covalently attached probe 4-nitrophenylanthranilate (NPA) with the capped NPs confirm the attachment of the NPA ligands to the surface functional group (-OH) of the citrate ligand. The FRET of a DNA base mimic, 2-aminopurine (2AP), with the NPs confirms the surface adsorption of 2AP. Our study may find relevance in the study of the interaction of individual manganite NPs with drug/ligand molecules.     

Conformationally constrained PNA analogues: structural evolution toward DNA/RNA binding selectivity

Since its discovery 12 years ago, aminoethylglycyl peptide nucleic acid (aeg-PNA) has emerged as one of the successful DNA mimics for potential therapeutic and diagnostic applications. An important requisite for in vivo applications that has received inadequate attention is engineering PNA analogues for able discrimination between DNA and RNA as binding targets. Our approach toward this aim is based on structural preorganization of the backbone to hybridization-competent conformations to impart binding selectivity. This strategy has allowed us to design locked PNAs to achieve specific hybridization with DNA or RNA with aims to increase the binding strength without losing the binding specificity. This Account presents results of our rationale in design of different conformationally constrained PNA analogues, their synthesis, and evaluation of hybridization specificities.     

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.     

A PNA-DNA2 Triple-Helix Molecular Switch-Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells

Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA-DNA₂ triplex. Moreover, the cyanine dye DiSC₂(5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label-free colorimetric sensing platform for miRNAs from cancer cells by using a PNA-DNA₂ triple-helix molecular switch (THMS) and DiSC₂(5). This sensing platform can detect miRNA-21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS-mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource-limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.     

Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea‐Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc‐based solid‐phase peptide synthesis in combination with a “tea‐bag” approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo‐CVX‐5c. Cyclotides were synthesized in a single‐pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose‐based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell‐based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell‐based assays for rapid screening.     

Probing Local Folding Allows Robust Metal Sensing Based on a Na+-Specific DNAzyme

Fluorescent metal sensors based on DNA often rely on changes in end-to-end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side-by-side comparison of two signaling strategies on a Na⁺-specific DNAzyme that contained a Na⁺-binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2-aminopurine (2AP)-labeled substrate strand. Na⁺ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na⁺ binding and forming the duplex on the 5′-end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na⁺ binding was achieved with a five base-pair stem. The effect on the 3′-end is more continuous, and the stem needs to form first before Na⁺ can bind. With an optimized substrate binding arm, a FRET-based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na⁺ failed to show a distinct difference from that of Li⁺ or K⁺; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.     

Influence of Cleavage Site on Global Folding of an RNA‐Cleaving DNAzyme

8–17 is a DNAzyme with metal‐dependent endoribonuclease activity. Recently, a variant termed 8–17NG was reported as the first nucleic acid enzyme capable of cleaving all 16 dinucleotide junctions of RNA with rate enhancements ranging from 1000‐ to 1 000 000 000‐fold over background activity. We attributed this broad‐ranging cleavage efficiency to global folding of the DNAzyme. We sought to examine the influence of dinucleotides at the cleavage site of 8–17NG on global folding by using three‐color (3c) FRET. By comparing the folding of 8–17NG with all 16 possible dinucleotide junctions, we found all examined DNAzyme–substrate constructs adopted a two‐step folding process in the presence of Mn²⁺, which was consistent with previous metal‐induced folding studies of 8–17. Interestingly, Mn²⁺ titration experiments also suggest that the second folding step is dependent on dinucleotide identity: purine–purine junctions allowed 8–17NG to fold at lower concentrations than pyrimidine–pyrimidine linkages. This finding was corroborated by RNA cleavage assays, in which the largest improvement in cleavage yield was observed in pyrimidine–pyrimidine junctions when [Mn²⁺] was increased. Taken together, these results support the previously observed hierarchy of 8–17 activity for different cleavage sites. Complemented by earlier sequence and structure–function studies, this investigation allowed for the first detailed examination of crucial relationships between the structural influence and junction preferences of nucleic acid‐catalyzed RNA cleavage reactions.     

Base pairing at the abasic site in DNA duplexes and its application in adenosine aptasensors

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.