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.     

Molecular thermodynamics of LNA:LNA base pairs and the hyperstabilizing effect of 5′‐proximal LNA:DNA base pairs

Locked nucleic acids (LNAs) can greatly enhance duplex DNA stability, and are therefore creating opportunities to improve therapeutics, as well as PCR‐based disease and pathogen diagnostics. Realizing the full potential of LNAs will require better understanding of their contributions to duplex stability, and the ability to predict their hydridization thermodynamics. Melting thermodynamics data for a large set of diverse duplexes containing LNAs in one or both strands are presented. Those data reveal that LNAs, when present on both strands, can stabilize a duplex not only through direct interaction with their base‐pair partner, but also through nonlocal hyperstablization effects created by LNA:LNA base pairs and/or specific patterns of oppositely oriented LNA:DNA base pairs. The data are, therefore, used to extend a thermodynamic model previously developed in our lab to permit accurate prediction of melting temperatures for duplexes bearing LNA substitutions within both strands using a classic group‐contribution approach. © 2015 American Institute of Chemical Engineers AIChE J, 61: 2711–2731, 2015     

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.     

An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Detection of DNA hybridization by a pyrene-labeled polyelectrolyte prepared by ATRP

A novel pyrene-labeled polyelectrolyte (Py-PDMAEMA⁺) has been prepared by atom transfer radical polymerization (ATRP) for detection of DNA hybridization. The electrostatic and hydrophobic interactions between Py-PDMAEMA⁺ and ssDNA (hairpin or linear DNA) kept them forming a complex probe which could be used as a turn-off fluorescent sensor. The fluorescence intensity of the probe decreased upon adding the complementary strand, since the pyrene moiety could be intercalated into the duplex and thus the fluorescence quenching by nucleotide bases was strengthened. The proposed intercalation mode was confirmed by the circular dichroism spectra and the fluorescence quenching study with iodide. This pyrene-labeled polyelectrolyte combined with ssDNA may establish a novel fluorescence sensing system for DNA hybridization.     

Potent Triple Helix Stabilization by 5′,3′‐Modified Triplex‐Forming Oligonucleotides

Anthraquinone and pyrene analogues attached to the 3′ and/or 5′ termini of triplex‐forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid‐phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 °C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 °C at pH 7 even though they do not contain any triplexstabilizing base analogues. These studies have implications for the design of triplex‐forming oligonucleotides for use in biology and nanotechnology.     

Pyrene-modified unlocked nucleic acids: synthesis, thermodynamic studies, and fluorescent properties

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.     

High‐Fidelity Recognition of RNA: Solution Structure of a DNA:RNA Hybrid Duplex with a Molecular Cap

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson–Crick pairing at their termini. Here we report the three‐dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5′ terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard‐to‐suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high‐fidelity probes for RNA.     

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.     

Discrimination of single-nucleotide alterations by G-specific fluorescence quenching

A new strategy for the detection of single-base alterations through fluorescence quenching by guanine (G) is described. We have devised a novel base-discriminating fluorescent (BDF) nucleoside, 4'PyT, that contains a pyrenecarboxamide fluorophore at the thymidine sugar's C4'-position. 4'PyT-containing oligodeoxynucleotides only exhibited enhanced fluorescence in response to the presence of a complementary adenine base. In contrast, the fluorescence of mismatched duplexes containing 4'PyT/N base pairs (N = C, G, or T) was considerably weaker. This highly A-selective fluorescence was a product of guanine-specific quenching efficiency; when the complementary base to 4'PyT was a mismatch, the pyrenecarboxamide fluorophore was able to interact intimately with neighboring G bases (the most likely interaction in the case of intercalation), so effective quenching by the G bases occurred in the mismatched duplexes. In contrast, duplexes containing 4'PyT/A base pairs exhibited strong emission, since in this case the fluorophores were positioned in the minor groove and able to escape fluorescence quenching by the G bases.     

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

Janus‐type nucleosides are heterocycles with two faces, each of which is designed to complement the H‐bonding interactions of natural nucleosides comprising a canonical Watson–Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence‐specific DNA recognition through the formation of J‐loops that will be more stable than D‐loops, which simply replaces one base‐pair with another. Herein, we report the synthesis of a novel Janus‐AT nucleoside analogue, J_AT, affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single J_AT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV‐melting and circular dichroism data demonstrated that within the context of a standard duplex, J_AT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the J_AT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H‐bonding. Finally, molecular dynamics simulations validated the observation that J_AT can substitute more effectively as an A‐analogue than as a T‐analogue without substantial distortion of the B‐helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A–T base pairs in DNA, and will lead to the development of oligo‐Janus‐nucleotide strands for sequence‐specific DNA recognition.     

1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA

In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.     

Locked nucleic acids and intercalating nucleic acids in the design of easily denaturing nucleic acids: thermal stability studies

Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.     

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.     

Fluorescent 2‐Aminopyridine Nucleobases for Triplex‐Forming Peptide Nucleic Acids

Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex‐forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2‐aminopyridine (M) nucleobase to a G‐C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3‐(1‐phenylethynyl)‐M and phenylpyrrolo‐M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple‐helix formation with cognate double‐stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3‐(1‐phenylethynyl)‐M may be used as a fluorescent nucleobase to study PNA–RNA triple‐helix formation.     

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable.     

7‐Azidomethoxy‐Coumarins as Profluorophores for Templated Nucleic Acid Detection

Templated nucleic acid detection is an emerging bioanalytical method that makes use of the target DNA or RNA strand to initiate a fluorogenic reaction. The Staudinger reduction holds particular promise for templated sensing of nucleic acids because the involved functional groups are highly chemoselective. Here, the azidomethoxy group, which can be removed under Staudinger conditions, is used to cage 7‐hydroxycoumarin fluorophores. Reduction by phosphines and subsequent loss of the azidomethoxy substituent induce a significant bathochromic shift of the major absorbance band in the near UV region. When excited at the appropriate wavelength, this change in the absorbance spectrum translates into a substantial fluorescence turn‐on signal. The described profluorophores are readily conjugated to amino‐modified DNAs and are rapidly uncaged by a triphenylphosphine–DNA probe under the control of a DNA template. In addition, turnover of the probes on the target strand occurs and yields substantial signal amplification.     

Developing an Updated Strategy for Estimating the Free-Energy Parameters in RNA Duplexes

For the last 20 years, it has been common lore that the free energy of RNA duplexes formed from canonical Watson–Crick base pairs (bps) can be largely approximated with dinucleotide bp parameters and a few simple corrective constants that are duplex independent. Additionally, the standard benchmark set of duplexes used to generate the parameters were GC-rich in the shorter duplexes and AU-rich in the longer duplexes, and the length of the majority of the duplexes ranged between 6 and 8 bps. We were curious if other models would generate similar results and whether adding longer duplexes of 17 bps would affect the conclusions. We developed a gradient-descent fitting program for obtaining free-energy parameters—the changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS), and the melting temperature (Tm)—directly from the experimental melting curves. Using gradient descent and a genetic algorithm, the duplex melting results were combined with the standard benchmark data to obtain bp parameters. Both the standard (Turner) model and a new model that includes length-dependent terms were tested. Both models could fit the standard benchmark data; however, the new model could handle longer sequences better. We developed an updated strategy for fitting the duplex melting data.