Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids.

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

Oligonucleotides Containing Phenoxazine Artificial Nucleobases: Triplex-Forming Abilities and Fluorescence Properties.

1,3-Diaza-2-oxophenoxazine ("phenoxazine"), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π-π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

2'-O,4'-C-Methylene-Bridged Nucleic Acids Stabilize Metal-Mediated Base Pairing in a DNA Duplex.

The 2'-O,4'-C-methylene-bridged or locked nucleic acid (2',4'-BNA/LNA), with an N-type sugar conformation, effectively improves duplex-forming ability. 2',4'-BNA/LNA is widely used to improve gene knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal-mediated base pairs (MMBPs), such as thymine (T)-HgII -T and cytosine (C)-AgI -C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2',4'-BNA/LNA in the field of MMBPs. 2',4'-BNA/LNA with 5-methylcytosine stabilized the MMBP of C with AgI ions. Moreover, the 2',4'-BNA/LNA sugar significantly improved the duplex-forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex-containing MMBPs. The results indicate that 2',4'-BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies.

A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9 and evaluation of antisense-mediated exon skipping.

Oligonucleotide-mediated splicing modulation is a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Recently, eteplirsen, a phosphorodiamidate morpholino oligomer-based splice-switching oligonucleotide (SSO) targeting DMD exon 51, was approved by the U.S. Food and Drug Administration as the first antisense-based drug for DMD patients. For further exploring SSOs targeting other exons in the DMD gene, the efficacy of exon skipping and protein rescue with each SSO sequence needs evaluations in vitro. However, only a few immortalized muscle cell lines derived from DMD patients have been reported and are available to test the efficacy of exon skipping in vitro. To solve this problem, we generated a novel immortalized DMD muscle cell line from the human rhabdomyosarcoma (RD) cell line. We removed DMD exons 51-57 (~0.3 Mb) in the RD cell line using the CRISPR/Cas9 system. Additionally, in this DMD model cell line, we evaluated the exon 50 skipping activity of previously reported SSOs at both the mRNA and protein levels. CRISPR/Cas9-mediated gene editing of the DMD gene in the RD cell line will allow for assessment of SSOs targeting most of the rare mutations in the DMD gene.

Mechanisms for removal of p-nitrophenol from aqueous solution using zero-valent iron.

Batch experiments were conducted to examine mechanisms for removal of p-nitrophenol (PNP) from aqueous solution using zero-valent iron (ZVI). Removal of PNP using ZVI was mainly attributed to three mechanisms: degradation, precipitation and adsorption. A complete removal of 30 mg L(-1) PNP with ZVI dosage of 1000 mg L(-1) achieved within 30 min at pH 3. The PNP removal rate in the acidic solutions was significantly suppressed at higher pH. The modified Langmuir-Hinshelwood kinetic model could successfully describe the PNP removal process using ZVI at different pH conditions. Total organic carbon (TOC) removal efficiencies were found to be almost independent of pH. While the TOC removal at lower pH was profoundly affected by the reductive and/or oxidative degradation, the adsorption was favorable at higher pH. The effect of dissolved oxygen on PNP removal was investigated at pH 3 where a maximum contribution of oxidative degradation could be expected. The PNP removal in the anoxic system purged with nitrogen gas was quick as well as that in the system being open to the air. However, the TOC removal under the anoxic condition was negligible as compared with that in the oxic system. The profiles of the intermediates formed during the PNP degradation indicated that the reductive degradation was predominant in the initial phase of the removal and subsequently the oxidative degradation occurred.

Enhancement of exon skipping activity by reduction in the secondary structure content of LNA-based splice-switching oligonucleotides.

PAGE and UV melting analysis revealed that longer LNA-based splice-switching oligonucleotides (SSOs) formed secondary structures by themselves, reducing their effective concentration. To avoid such secondary structure formation, we introduced 7-deaza-2'-deoxyguanosine or 2'-deoxyinosine into the SSOs. These modified SSOs, with fewer secondary structures, showed higher exon skipping activities.