Identification of the Myogenetic Oligodeoxynucleotides (myoDNs) That Promote Differentiation of Skeletal Muscle Myoblasts by Targeting Nucleolin

Abstract

Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13–15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.

Myogenetic Oligodeoxynucleotide (myoDN) Recovers the Differentiation of Skeletal Muscle Myoblasts Deteriorated by Diabetes Mellitus

“…All phosphodiester bonds of iSN04 (5′-AGA TTA GGG TGA GGG TGA-3′) were phosphorothioated to increase resistance to nucleases. Phosphorothioated iSN04 was synthesized and HPLC-purified (GeneDesign, Osaka, Japan), then was dissolved in endotoxin-free water as previously reported ( Shinji et al, 2021 ). Palmitic acid (Wako, Osaka, Japan), which is the most abundant (occupying 20–30%) saturated fatty acid in human ( Gesteiro et al, 2019 ), was dissolved in chloroform to prepare a high concentration stock (600 mM) to decrease treatment volume to myoblasts ( Aguer et al, 2010 ).…”

“…The murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) was maintained in a growth medium for C2C12 cells (C2-GM) consisting of DMEM (Nacalai, Osaka, Japan) with 10% fetal bovine serum and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (PS) (Nacalai). hMBs and C2C12 cells were differentiated in a differentiation induction medium (DIM) consisting of DMEM with 2% horse serum (HyClone; GE Healthcare, Salt Lake City, UT, United States) and PS ( Nihashi et al, 2019b ; Shinji et al, 2021 ).…”

“…Optimal iSN04 concentration for each MB was defined as indicating the highest ratio of MHC + cells without affecting cell number because the sensitivities to iSN04 differ among MBs. Immunocytochemistry of myoblasts was performed as previously described ( Takaya et al, 2017 ; Nihashi et al, 2019a ; Shinji et al, 2021 ). The myoblasts were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with 0.5 μg/ml mouse monoclonal anti-MHC antibody (MF20; R&D Systems, Minneapolis, MN, United States) and 1.0 μg/ml rabbit polyclonal anti-nucleolin antibody (ab22758; Abcam, Cambridge, United Kingdom).…”

“…We recently identified myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand nucleotides that promote myoblast differentiation ( Nihashi et al, 2020 ; Shinji et al, 2021 ). One of the myoDNs, iSN04, is directly incorporated into myoblasts and serves as an aptamer that physically interacts with nucleolin ( Shinji et al, 2021 ). Nucleolin has been known to target the untranslated region of p53 mRNA to interfere with its translation ( Takagi et al, 2005 ; Chen et al, 2012 ).…”

“…Nucleolin has been known to target the untranslated region of p53 mRNA to interfere with its translation ( Takagi et al, 2005 ; Chen et al, 2012 ). In myoblasts, iSN04 antagonizes nucleolin, rescues p53 protein levels, and eventually facilitates myotube formation ( Shinji et al, 2021 ). In this study, we aimed to determine that iSN04 recovers the deteriorated differentiation of myoblasts isolated from patients with DM.…”