Keiji Murayama

Reversible photoswitching of RNA hybridization at room temperature with an azobenzene C-nucleoside.

Photoregulation of RNA remains a challenging task as the introduction of a photoswitch entails changes in the shape and the stability of the duplex that strongly depend on the chosen linker strategy. Herein, the influence of a novel nucleosidic linker moiety on the photoregulation efficiency of azobenzene is investigated. To this purpose, two azobenzene C-nucleosides were stereoselectively synthesized, characterized, and incorporated into RNA oligonucleotides. Spectroscopic characterization revealed a reversible and fast switching process, even at 20 °C, and a high thermal stability of the respective cis isomers. The photoregulation efficiency of RNA duplexes upon trans-to-cis isomerization was investigated by using melting point studies and compared with the known D-threoninol-based azobenzene system, revealing a photoswitching amplitude of the new residues exceeding 90 % even at room temperature. Structural changes in the duplexes upon photoisomerization were investigated by using MM/MD calculations. The excellent photoswitching performance at room temperature and the high thermal stability make these new azobenzene residues promising candidates for in-vivo and nanoarchitecture photoregulation applications of RNA.

Unexpectedly Stable Artificial Duplex from Flexible Acyclic Threoninol

A new foldamer, acyclic threoninol nucleic acid (aTNA), has been synthesized by tethering each of the genetic nucleobases A, G, C, and T to d-threoninol molecules, which were then incorporated as building blocks into a scaffold bearing phosphodiester linkages. We found that with its fully complementary strand in an antiparallel fashion, the aTNA oligomer forms an exceptionally stable duplex that is far more stable than corresponding DNA or RNA duplexes, even though single-stranded aTNA is rather flexible and thus does not take a preorganized structure.

Control of the Chirality and Helicity of Oligomers of Serinol Nucleic Acid (SNA) by Sequence Design

The construction of an artificial double helix that mimics natural DNA or RNA has been one of the most challenging endeavors in chemistry. Recently, Meggers and co-workers showed that even a simple acyclic propylene glycol with two carbon atoms in the main chain (see (S)-GNA in Figure 1) as a nucleobase tether could form a more stable duplex than that of native DNA or RNA. This pioneering work prompted us to synthesize a new foldamer with three carbon atoms in the main chain, acyclic threoninol nucleic acid (aTNA), from d-threoninol. We found that aTNA has the following properties: 1) duplex formation involves complementary pairing in an antiparallel fashion, as for natural DNA or RNA; 2) owing to the flexibility of the backbone, the singlestranded state does not adopt a characteristic preorganized structure; 3) the thermal stability of the duplex is far greater than that of the natural DNA or RNA duplex and even higher than that of the GNA duplex. Studies on aTNA as well as GNA and peptide nucleic acid (PNA) 16] have confirmed that scaffold rigidity is not a prerequisite for stable duplex formation as previously thought. However, unlike PNA, with an acyclic scaffold, aTNA can not cross-hybridize with either natural DNA or natural RNA. Although A15 of (S)-GNA can hybridize with U15, the incorporation of several GC pairs severely destabilizes the duplex with RNA. Thus, there are no artificial nucleic acids comprising a fully acyclic backbone with a phosphodiester linkage that can cross-hybridize with DNA or RNA without sequence limitation. We hypothesize that the threoninol scaffold is still not flexible enough to form a duplex with natural DNA or RNA. Herein, we propose a new artificial nucleic acid, serinol nucleic acid (SNA, see Figure 1a), with a 2-amino-1,3propanediol (serinol) scaffold, which is even more flexible than threoninol. In comparison with aTNA (Figure 1a), the only structural difference is the lack of a methyl group next to the amino group. However, this small change affords the SNA oligomer a unique stereochemical property: since this methyl group provides chirality, its absence makes the scaffold achiral as well as flexible. Accordingly, the chirality of the “pure” SNA oligomer synthesized from four SNA monomers (or the helicity of its duplex) depends only on its sequence (see below). This property is specific to the SNA oligomer; DNA, RNA, and previously synthesized aTNA all have chirality (or helicity) that is inherently determined by the chirality of the scaffold. In the present study, we first demonstrated this unique stereochemical property and then cross-hybridized the SNA oligomer, which was found to recognize both DNA and RNA sequence specifically. The chemical structure of the SNA oligomer is shown in Figure 1a. Serinol (2-amino-1,3-propanediol), which, like DNA, has three carbon atoms in its backbone, is an achiral diol. However, modification of the two hydroxy groups with different functional groups to form an SNA monomer (FigFigure 1. a) Chemical structures of DNA and SNA. S and R termini were named according to the chirality of the terminal residue. The SNA monomer for the DNA synthesizer is also shown (DMT= dimethoxytrityl). b) The mirror image of SNA with an asymmetrical sequence ((S)-AT-(R)) is identical to SNA with the reverse sequence ((S)-TA-(R)). c) SNA with a symmetrical sequence ((S)-TT-(R)) is identical to its mirror image.

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA≫RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D-threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross-hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15-mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.

Enhancement of Stability and Activity of siRNA by Terminal Substitution with Serinol Nucleic Acid (SNA)

RNA interference (RNAi ), sequence‐specific gene silencing triggered by double‐stranded, small interfering RNA (siRNA), has become a facile and effective tool for biological research and holds potential for therapeutic applications. However, the application of siRNA is hindered by susceptibility to nucleases and off‐target effects. In this study, we introduced artificial nucleotides, serinol nucleic acid (SNA), with an acyclic scaffold, at the termini of siRNA strands. Our aim was appropriately to accommodate the antisense strand in an RNA‐induced silencing complex (RISC) by inhibiting sense‐strand incorporation and thus improve resistance to nuclease‐mediated degradation. Substitution of SNA into siRNA at both termini of the sense strand and at the 3′ terminus of the antisense strand improved antisense strand selectivity remarkably in the formation of RISC, RNAi activity, and nuclease resistance.

Azobenzene C-Nucleosides for Photocontrolled Hybridization of DNA at Room Temperature.

Herein, we report the reversible light-regulated destabilization of DNA duplexes by using azobenzene C-nucleoside photoswitches. The incorporation of two different azobenzene residues into DNA and their photoswitching properties are described. These new residues demonstrate a photoinduced destabilization effect comparable to the widely applied D-threoninol-linked azobenzene switch, which is currently the benchmark. The photoswitches presented herein show excellent photoswitching efficiencies in DNA duplexes - even at room temperature - which are superior to commonly used azobenzene-based nucleic acid photoswitches. In addition, these photoswitching residues exhibit high thermal stability and excellent fatigue resistance, thus rendering them one of the most efficient candidates for the regulation of duplex stability with light.

Visible-Light-Triggered Cross-Linking of DNA Duplexes by Reversible [2+2] Photocycloaddition of Styrylpyrene.

Reversible photo-cross-linking of a DNA duplex through the [2+2] photocycloaddition of styrylpyrene is reported. Styrylpyrene moieties on d-threoninol linkers were introduced into complementary positions on DNA strands. Irradiation of the styrylpyrene pair in the duplex with visible light at λ=455 nm induced a [2+2] photocycloaddition between styrylpyrenes that cross-linked the two strands of the duplex. Two diastereomers were formed after [2+2] photocycloaddition as a result of rotation of the styrylpyrene residues. Also, the cycloreversion reaction was induced by UV light at λ=340 nm, which reversibly yielded the uncross-linked strands.

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.

A “sugar-deficient” G-quadruplex: incorporation of aTNA in G4 structures

The effects of modification of the phosphodiester backbone or the guanine bases on G-quadruplex formation have been widely investigated. Only a few studies have investigated the effects of deoxyribose or ‘sugar’ modifications on G-quadruplex structure. Here, we evaluated the structural, thermodynamic, and kinetic properties of the parallel quadruplexes formed by the sequence d(TGGGGT) in which each guanine base was substituted, one at a time, with acyclic threoninol nucleic acid (aTNA). We found that all sequences were able to form G-quadruplexes; however, the presence of aTNA resulted in the formation of a mixture of quadruplex structures in some cases. Furthermore, the presence of a single substitution at any position resulted in destabilization of the G-quadruplex relative to that formed by the unmodified sequence. The introduction of the aTNA in terminal quartets was the most detrimental to stability. In addition, kinetic experiments showed that, compared to its unmodified counterpart sequence d(TGGGGT), the substitution of a normal guanine nucleotide by aTNA decelerated quadruplex formation except when the aTNA was at the 5′ most guanine of the sequence. In summary, our studies indicate that the deoxyribose sugar affects the properties of G-quadruplex structures.

Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in mdx myotubes

Serinol nucleic acid (SNA) is a novel nucleic acid analogue that can form highly stable heteroduplexes with complementary DNA and RNA sequences. Structurally, SNA is a close mimic to peptide nucleic acid (PNA) which is widely used in diagnostic and therapeutic applications. SNA chemistry is relatively new, and so far the scope of SNA has only been explored in improving the efficacy of small interfering RNA and for developing a highly sensitive molecular beacon for diagnostic applications. In this study, we investigated the potential of SNA-modified antisense oligonucleotide (AO) in parallel to PNA-oligo for splice-modulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20-mer SNA and PNA antisense oligonucleotide (AO) designed to induce exon-23 skipping in the mouse dystrophin gene transcript. Our results demonstrated that the SNA AO induced exon-23 skipping at all tested concentrations, whereas the corresponding PNA AO failed to induce any exon-23 skipping upon 24 hours of transfection using Lipofectin transfection reagent. Our results further expands the potential of SNA oligonucleotides in therapeutic applications.