Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.     

Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking

DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole (^CNVK) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy.     

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

A Single‐Electrode,Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc–PNA biosensor (Fc: ferrocenyl, C₁₀H₉Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc–PNA capture probes were therefore synthesized by N‐terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi‐reversible redox processes of the Fc^0/+ redox couple with a significant difference in their electrochemical half‐wave potentials of ΔE_1/2=160 mV. A carefully designed biosensor interface, consisting of a ternary self‐assembled monolayer (SAM) of the two C‐terminal cysteine‐tethered Fc–PNA capture probes and 6‐mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear‐cut electrochemical signal about the hybridization event. The discrimination of single‐nucleotide polymorphism (SNP) was proven with a chosen single‐mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual‐potential sensor under real‐life conditions.     

Peptide Nucleic Acid Conjugated with Ruthenium‐Complex Stabilizing Double‐Duplex Invasion Complex Even under Physiological Conditions

Peptide nucleic acid (PNA) can form a stable duplex with DNA, and, accordingly, directly recognize double‐stranded DNA through the formation of a double‐duplex invasion complex, wherein a pair of complementary PNA strands form two PNA/DNA duplexes. Because invasion does not require prior denaturation of DNA, PNA holds great potential for in cellulo or in vivo applications. To broaden the applicability of PNA invasion, we developed a new conjugate of PNA with a ruthenium complex. This Ru–PNA conjugate exhibits higher DNA‐binding affinity, which results in enhanced invasion efficiency, even under physiological conditions.     

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.     

As fast and selective as enzymatic ligations: unpaired nucleobases increase the selectivity of DNA-controlled native chemical PNA ligation

DNA-controlled reactions offer interesting opportunities in biological, chemical, and nanosciences. In practical applications, such as in DNA sequence analysis, the sequence fidelity of the chemical-ligation reaction is of central importance. We present a ligation reaction that is as fast as and much more selective than enzymatic T4 ligase-mediated oligonucleotide ligations. The selectivity was higher than 3000-fold in discriminating matched from singly mismatched DNA templates. It is demonstrated that this enormous selectivity is the hallmark of the particular ligation architecture, which is distinct from previous ligation architectures designed as "nick ligations". Interestingly, the fidelity of the native chemical ligation of peptide nucleic acids was increased by more than one order of magnitude when performing the ligation in such a way that an abasic-site mimic was formed opposite an unpaired template base. It is shown that the high sequence fidelity of the abasic ligation could facilitate the MALDI-TOF mass-spectrometric analysis of early cancer onset by allowing the detection of as little as 0.2 % of single-base mutant DNA in the presence of 99.8 % wild-type DNA.     

The Inhibition Effect of Photo-Cross-Linking between Probes in Photo-Induced Double Duplex Invasion DNA

Double duplex invasion (DDI) DNA is a useful antigene method that inhibits expression of genomic DNA. We succeeded in performing photoinduced-DDI (pDDI) using ultrafast photo-cross-linking. 5-Cyanouracil (^CNU) has been used in pDDI to inhibit photo-cross-linking between probes, but its importance has not been clarified. Therefore, in this study, we evaluated the effect of spacer (S) and d-spacer (dS) that exhibit photo-cross-linking ability similar to that of ^CNU. ^CNU exhibited the highest pDDI efficiency, and S, dS, and T were not very different. The photo-cross-linking inhibitory effect was better with S and dS than with thymidine (T). Conversely, the thermal stability was significantly lower with S and dS than with T. The results suggest that the pDDI efficiency is determined by the balance between the photo-cross-linking inhibitory effect and the thermal stability, which is the introduction efficiency for double-stranded DNA. Therefore, ^CNU, which has a photo-cross-linking inhibitory effect and a high T_m value, showed the highest inhibitory efficiency.     

Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA

Fluorescence techniques for the investigation of biomolecules and their folding pathways require an efficient labeling strategy. A common method to internally label large RNAs involves the introduction of long loops for hybridization of fluorophore‐carrying DNA strands. Such loops often disturb the structure, and thus the functionality, of the RNA. Here we show, in a proof of concept study with a >600 nucleotide group II intron ribozyme, that the usage of the nucleic acid analogue peptide nucleic acid (PNA) is more efficient in several aspects, minimizing the required structural modifications of the RNA. We demonstrate by various methods, including smFRET, that much smaller concentrations and shorter PNAs can be applied, compared to DNA, for rapid and specific internal RNA labeling. The folding pathway and catalytic activity of this large ribozyme is only minimally affected by the PNA, but the background signal is significantly reduced.     

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA‐templated ligation includes easy on‐resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5‐dimethoxy‐2‐nitrobenzaldehyde‐based linker that allowed the photocleavable linkage of two bio‐oligomers was developed.     

PNA Molecular Beacons Assembled by Post‐Synthetic Click Chemistry Functionalization

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post‐synthetic on‐resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide‐containing monomers into the oligomer by automatic solid‐phase peptide synthesis and subsequent derivatization with pyrene moieties by copper‐catalyzed azide–alkyne cycloaddition. Two pyrene‐based quencher‐free PNA molecular beacons, a stemless MB and one possessing a stem–loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem–loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.     

DNA as a Platform to Program Assemblies with Emerging Functions in Chemical Biology

The programmability of oligonucleotide recognition offers an attractive platform for directing the assembly of ligands that can interact cooperatively with a target or the assembly of reactive partner that can engage in chemical reactions. In both cases, an oligonucleotide directs the assembly, which yields a functional output. In terms of the controlled display of ligands, several examples have shown a significant increase in binding upon ligand assembly using small molecule fragments, peptides, glycans, and protein fragments. Combinatorial approaches to identifying the optimal pairing have also been reported. In terms of nucleic acid triggered reactions, several robust chemistries have been reported, leading to the unmasking of different fluorophores or bioactive molecules as well as the synthesis of a bioactive compound in response to DNA or even cellular RNA. Herein we present the historical context of this work and summarize recent developments in this area.