Unexpectedly stable homopurine parallel triplex of SNA:RNA*SNA and L-aTNA:RNA*L-aTNA.

Homopurine strands are known to form antiparallel triplexes stabilized by G*G and A*A Hoogsteen pairs, which have two hydrogen bonds. But there has been no report on the parallel triplex formation of homopurine involving both adenosine and guanosine to the duplex. In this paper, we first report parallel triplex formation between a homopurine serinol nucleic acid (SNA) strand and an RNA/SNA duplex. Melting profiles revealed that the parallel SNA:RNA*SNA triplex was remarkably stable, even though the A*A pair has a single hydrogen bond. An L-acyclic threoninol nucleic acid (L-aTNA) homopurine strand also formed a stable parallel triplex with an L-aTNA/RNA duplex.

Illuminating miRNA Inhibition: Visualizing the Interaction between Anti-miRNA Oligonucleotide and Target miRNA Using FRET.

Anti-miRNA oligonucleotides (anti-miRs) effectively and specifically inhibit the function of individual miRNAs and have the potential to serve as a novel class of nucleic acid therapeutic. However, the details of the mechanisms of anti-miRs in cells have not yet been clarified sufficiently. In particular, the localization of the complexes of anti-miRs and target miRNA in cells remains unclear. We previously developed anti-miRs composed of serinol nucleic acid (SNA) that very effectively inhibited miRNA-mediated silencing activity. Here we describe an imaging system based on the fluorescence resonance energy transfer (FRET) designed by miRNAs labeled with fluorophore-quencher pairs and an SNA-based anti-miR labeled with an acceptor dye. We discovered that the anti-miR hybridizes with the miRNA in the miRNA-induced silencing complex (miRISC), which is the active complex formed by miRNA and Ago2 in cells within P-bodies. Based on FRET ratio analysis, we hypothesize that the complex formed by the anti-miR and the miRNA in P-bodies is dynamic, with anti-miR complexing the miRISC, followed by miRNA release and degradation. Our findings provide valuable insights into the mechanism of action of anti-miRs and enable further studies of miRNA-targeted therapeutics.

Syntheses of Base-Labile Pseudo-Complementary SNA and l-aTNA Phosphoramidite Monomers.

We previously synthesized phosphoramidite monomers bearing Boc-protected 2,6-diaminopurine (D) and 2-methyl-4-methoxybenzyl-protected 2-thiouracil (sU) as building blocks for the preparation of pseudo-complementary serinol nucleic acids (SNAs). Since SNA is stable under acidic conditions, an acid-deprotection step could be inserted into the work-up. However, as the 4,4'-dimethoxytrityl group was concurrently removed at this step, purification of SNA by reversed-phase HPLC was difficult. Here, we report the syntheses of SNA and acyclic l-threoninol nucleic acid (l-aTNA) phosphoramidite monomers with bis(phenoxyacetyl)-protected D and 4-acetoxybenzyl-protected sU, both of which can be deprotected under mild basic conditions. Using these monomers, we prepared pseudo-complementary SNA and l-aTNA in high yield using conventional oligonucleotide synthesis protocols. These monomers can be used for large-scale syntheses of SNAs and l-aTNAs.

Xeno nucleic acids (XNAs) having non-ribose scaffolds with unique supramolecular properties.

DNA and RNA have significance as genetic materials, therapeutic potential, and supramolecular properties. Advances in nucleic acid chemistry have enabled large-scale synthesis of DNA and RNA oligonucleotides and oligomers of non-natural nucleic acids, including artificial nucleic acids (xeno nucleic acids; XNAs) with non-ribose scaffolds. In this feature article, we review the chemical structures of XNAs with non-ribose scaffolds, their hybridization abilities, and their unique behaviors with a particular focus on the acyclic XNAs. First, we overview XNAs with non-ribose cyclic scaffolds and then those with acyclic scaffolds by focusing on their hybridization abilities with themselves and with DNA and RNA, and discuss the unexpectedly stable homo-duplex formation of acyclic XNAs. Next, we shed light on our acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) and show their helical preferences based on their chirality, then orthogonal control of hybridization and helical amplification of achiral XNAs are demonstrated. Finally, we show non-enzymatic template-directed synthesis of L-aTNA, and the creation of an artificial genetic system with XNAs with non-ribose scaffolds is described as a future prospect.

Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing Intermediates That Are Generated in Cells.

microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cytosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the subsequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer- or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demonstrated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.

Investigation of Strand-Selective Interaction of SNA-Modified siRNA with AGO2-MID.

Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5'-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5' end of the passenger strand.

Crystallographic snapshots of the EF-hand protein MCFD2 complexed with the intracellular lectin ERGIC-53 involved in glycoprotein transport.

The transmembrane intracellular lectin ER-Golgi intermediate compartment protein 53 (ERGIC-53) and the soluble EF-hand multiple coagulation factor deficiency protein 2 (MCFD2) form a complex that functions as a cargo receptor, trafficking various glycoproteins between the endoplasmic reticulum (ER) and the Golgi apparatus. It has been demonstrated that the carbohydrate-recognition domain (CRD) of ERGIC-53 (ERGIC-53CRD) interacts with N-linked glycans on cargo glycoproteins, whereas MCFD2 recognizes polypeptide segments of cargo glycoproteins. Crystal structures of ERGIC-53CRD complexed with MCFD2 and mannosyl oligosaccharides have revealed protein-protein and protein-sugar binding modes. In contrast, the polypeptide-recognition mechanism of MCFD2 remains largely unknown. Here, a 1.60 Šresolution crystal structure of the ERGIC-53CRD-MCFD2 complex is reported, along with three other crystal forms. Comparison of these structures with those previously reported reveal that MCFD2, but not ERGIC-53-CRD, exhibits significant conformational plasticity that may be relevant to its accommodation of various polypeptide ligands.

Designer Biopolymers: Self-Assembling Proteins and Nucleic Acids.

Nature has evolved sequence-controlled polymers such as DNA and proteins over its long history [...].

A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity.

A triplex-forming oligonucleotide (TFO) linear probe containing perylene derivatives was synthesized. The TFO linear probe formed a remarkably stable triplex with a target DNA duplex, resulting in the light-up of fluorescence emission. The sensitivity was extremely high even at pH 7. Detection of PCR-amplified target DNA was demonstrated.

Improved secretion of glycoproteins using an N-glycan-restricted passport sequence tag recognized by cargo receptor.

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.

Incorporation of Pseudo-complementary Bases 2,6-Diaminopurine and 2-Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization.

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.

Intrastrand backbone-nucleobase interactions stabilize unwound right-handed helical structures of heteroduplexes of L-aTNA/RNA and SNA/RNA.

Xeno nucleic acids, which are synthetic analogues of natural nucleic acids, have potential for use in nucleic acid drugs and as orthogonal genetic biopolymers and prebiotic precursors. Although few acyclic nucleic acids can stably bind to RNA and DNA, serinol nucleic acid (SNA) and L-threoninol nucleic acid (L-aTNA) stably bind to them. Here we disclose crystal structures of RNA hybridizing with SNA and with L-aTNA. The heteroduplexes show unwound right-handed helical structures. Unlike canonical A-type duplexes, the base pairs in the heteroduplexes align perpendicularly to the helical axes, and consequently helical pitches are large. The unwound helical structures originate from interactions between nucleobases and neighbouring backbones of L-aTNA and SNA through CH-O bonds. In addition, SNA and L-aTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA. The unique structural features of the RNA-recognizing mode of L-aTNA and SNA should prove useful in nanotechnology, biotechnology, and basic research into prebiotic chemistry.

Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution.

Although DNA can form triplex and quadruplex structures through hydrogen bonds, design and preparation of structures with more than five strands is difficult even when artificial nucleic acids are used. Herein we report a hexaplex formed by oligomers of artificial nucleic acids bearing bifacial molecules on d-threoninol. Aminopyrimidine and cyanuric acid derivatives were selected as bases because they have complementary hydrogen bonding patterns. The complex formed by aminopyrimidine and cyanuric acid decamers melted with large hysteresis. Hexaplex formation was indicated by gel electrophoresis, size exclusion chromatography and atomic force microscopy imaging, and proven directly through native mass spectrometry. CD measurements and molecular dynamics simulations indicated that the hexaplex adopts a helical structure. The hexaplex formation was highly dependent on pH and the presence of divalent cations. The hexaplex was stable in aqueous solution, and its unique structure and properties may lead to novel nanostructures, molecular assemblies, metal sensors, and ion channels.

Development of Visible-Light-Responsive RNA Scissors Based on a 10-23 DNAzyme.

The 10-23 DNAzyme is an artificially developed functional oligonucleotide that can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme incorporating a photoresponsive DNA overhang complementary to the catalytic core region. The photoresponsive overhang region of the DNAzyme included either azobenzene components (Azos) or 2,6-dimethyl-4-(methylthio)azobenzene units (SDM-Azos) each attached to a d-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photoresponsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. cis Isomerization of Azos or SDM-Azos, induced by 365 or 400 nm light, respectively, destabilized the duplex between the photoresponsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible photoswitching of the DNAzyme activity was achieved by use of specific light irradiation. Further, light-dependent photoswitching of protein expression in the presence of the DNAzyme was demonstrated. Thus, this photo-driven DNAzyme has potential for application as a photocontrolled gene silencing system and a photoactivatable gene expression system.

DNA Microcapsule for Photo-Triggered Drug Release Systems.

In this study we constructed spherical photo-responsive microcapsules composed of three photo-switchable DNA strands. These strands first formed a three-way junction (TWJ) motif that further self-assembled to form microspheres through hybridization of the sticky-end regions of each branch. To serve as the photo-switch, multiple unmodified azobenzene (Azo) or 2,6-dimethyl-4-(methylthio)azobenzene (SDM-Azo) were introduced into the sticky-end regions via a d-threoninol linker. The DNA capsule structure deformed upon trans-to-cis isomerization of Azo or SDM-Azo induced by specific light irradiation. In addition, photo-triggered release of encapsulated small molecules from the DNA microcapsule was successfully achieved. Moreover, we demonstrated that photo-triggered release of doxorubicin caused cytotoxicity to cultured cells. This biocompatible photo-responsive microcapsule has potential application as a photo-controlled drug-release system.

Introduction of 2,6-Diaminopurines into Serinol Nucleic Acid Improves Anti-miRNA Performance.

MicroRNAs (miRNAs) are endogenous small RNAs that regulate gene expression at the post-transcriptional level by sequence-specific hybridisation. Anti-miRNA oligonucleotides (AMOs) are inhibitors of miRNA activity. Chemical modification of AMOs is required to increase binding affinity and stability in serum and cells. In this study, we synthesised AMOs with our original acyclic nucleic acid, serinol nucleic acid (SNA), backbone and with the artificial nucleobase 2,6-diaminopurine. The AMO composed of only SNA had strong nuclease resistance and blocked endogenous miRNA activity. A significant improvement in anti-miRNA activity of the AMO was achieved by introduction of a 2,6-diaminopurine residues into the SNA backbone. In addition, we found that the enhancement in AMO activity depended on the position of the 2,6-diaminopurine residue in the sequence. The high potency of the SNA-AMOs suggests that these oligomers will be useful as therapeutic reagents for control of miRNA function in patients and as tools for investigating the roles of microRNAs in cells.

Dynamics of Inter-DNA Chain Interaction of Photoresponsive DNA.

Photoresponsive DNA modified with azobenzene is an attractive design molecule for efficient photoregulation of DNA hybridization, which may be used for controlling DNA functions. Although the essential step of photocontrolling DNA is the initial isomerization of the azobenzene, the dissociation/association kinetics remain unknown. Here, the time-resolved diffusion method was used to trace the dissociation/association processes of photoresponsive DNA. Although the isomerization of azobenzene occurs in picoseconds, the dissociation of the double-stranded DNA to single-stranded DNA triggered by the trans to cis isomerization takes place ∼10(7) times slower, with a time constant of 670 µs at 200 µM. From the concentration dependence, the dissociation and association rates were determined. Furthermore, the reaction rate from the single- to double-stranded DNA after the cis to trans isomerization was measured to be 3.6 ms at 200 µM. The difference in the melting temperatures of DNA between tethered trans- and cis-azobenzene is explained by the different rate of dissociation of the double-stranded form.

Strand-invading linear probe combined with unmodified PNA.

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration.

Isotope effect on the circular dichroism spectrum of methyl α-D-glucopyranoside in aqueous solution.

H/D isotope effect on the circular dichroism spectrum of methyl α-D-glucopyranoside in aqueous solution has been analyzed by multicomponent density functional theory calculations using the polarizable continuum model. By comparing the computational spectra with the corresponding experimental spectrum obtained with a vacuum-ultraviolet circular dichroism spectrophotometer, it was demonstrated that the isotope effect provides insights not only into the isotopic difference of the intramolecular interactions of the solutes, but also into that of the solute-solvent intermolecular interaction.

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.