Identification of a Novel Osteogenetic Oligodeoxynucleotide (osteoDN) That Promotes Osteoblast Differentiation in a TLR9-Independent Manner

Nihashi, Yuma; Miyoshi, Mana; Umezawa, Koji; Shimosato, Takeshi; Takaya, Tomohide

doi:10.3390/nano12101680

Cited by 10 publications

(10 citation statements)

References 53 publications

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“…Intracellular uptake of iSN04 without a carrier has been reported in myoblasts [5]. As shown in Figure 1B, 6-FAM-iSN04 was autonomously internalized into A10 cells within 30 min and diffused into the cytoplasm and nucleoplasm by 4 h, which is generally observed for oligodeoxynucleotide transport by gymnosis [5,20,27]. These results demonstrate that iSN04 as an anti-nucleolin aptamer is able to act intracellularly in A10 cells.…”

Section: Resultssupporting

confidence: 70%

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

Miyoshi,

Shimosato,

Takaya

2024

Preprint

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De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining VSMCs in a contractile differentiated state is therefore a promising therapeutic strategy for atherosclerosis. We have reported the 18-base myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes skeletal and myocardial differentiation. The present study investigated the effect of iSN04 on VSMCs because nucleolin has been reported to contribute to VSMC de-differentiation under pathophysiological conditions. Nucleolin was localized in the nucleoplasm and nucleoli of both rat and human VSMCs. iSN04 without carrier was spontaneously incorporated into VSMCs, indicating that iSN04 would serve as an anti-nucleolin aptamer. iSN04 treatment decreased the ratio of EdU+ proliferating VSMCs and increased the expression of α-smooth muscle actin, a contractile marker of VSMCs. iSN04 also suppressed angiogenesis of mouse aortic rings ex vivo, which is a model of pathological angiogenesis involved in plaque formation, growth, and rupture. These results demonstrate that antagonizing nucleolin with iSN04 preserves VSMC differentiation, providing a nucleic acid drug candidate for the treatment of vascular disease.

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Section: Resultssupporting

confidence: 70%

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

Miyoshi,

Shimosato,

Takaya

2024

Preprint

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“…PS-ODNs in which all phosphodiester bonds were phosphorothioated to enhance nuclease resistance in cell culture were synthesized and HPLC-purified (GeneDesign, Osaka, Japan) [7,16]. Unmodified oligonucleotides without phosphorothioate for nuclear magnetic resonance (NMR) and native polyacrylamide gel electrophoresis (PAGE) were synthesized and HPLC-purified (Hokkaido System Science, Sapporo, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The two molecules of iMyo01 (DNA) in explicit solvent were simulated by trivialtrajectory parallelization (TTP-) McMD method [7,16]. The simulation system (Figure S1) contained two iMyo01 molecules, 10,252 water molecules, 23 K + ions, and one Cl − ion to be neutralized electrically.…”

Section: Methodsmentioning

confidence: 99%

Development of the 12-Base Short Dimeric Myogenetic Oli-godeoxynucleotide That Induces Myogenic Differentiation

Umezawa,

Ikeda,

Sakamoto

et al. 2024

Preprint

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A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5'-TTG GGT GGG GAA-3'), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs.

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“…Phosphorothioated iSN04 (5′-AGA TTA GGG TGA GGG TGA-3′) [ 16 , 17 , 18 , 19 , 26 ] was synthesized (GeneDesign, Osaka, Japan) and purified by reversed-phase high performance liquid chromatography (RP-HPLC) using XBridge BEH C18 column (pore size 130 Å; particle size, 2.5 μm; analytical size, 4.6 × 75 mm) (Waters, Milford, MA, USA) at 60 °C under ion-pairing condition to guarantee >95% of purity. AS1411 (5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′), having a phosphodiester backbone, was synthesized and desalted (Integrated DNA Technologies, Coralville, IA, USA) [ 16 , 27 ].…”

Section: Methodsmentioning

confidence: 99%

Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells

et al. 2022

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Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10–30 μM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 μM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10–30 μM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

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Identification of a Novel Osteogenetic Oligodeoxynucleotide (osteoDN) That Promotes Osteoblast Differentiation in a TLR9-Independent Manner

Cited by 10 publications

References 53 publications

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

Development of the 12-Base Short Dimeric Myogenetic Oli-godeoxynucleotide That Induces Myogenic Differentiation

Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells

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