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

Dysfunction of bone-forming cells, osteoblasts, is one of the causes of osteoporosis. Accumulating evidence has indicated that oligodeoxynucleotides (ODNs) designed from genome sequences have the potential to regulate osteogenic cell fate. Such osteogenetic ODNs (osteoDNs) targeting and activating osteoblasts can be the candidates of nucleic acid drugs for osteoporosis. In this study, the ODN library derived from the Lacticaseibacillus rhamnosus GG genome was screened to determine its osteogenetic effect on murine osteoblast cell line MC3T3-E1. An 18-base ODN, iSN40, was identified to enhance alkaline phosphatase activity of osteoblasts within 48 h. iSN40 also induced the expression of osteogenic genes such as Msx2, osterix, collagen type 1α, osteopontin, and osteocalcin. Eventually, iSN40 facilitated calcium deposition on osteoblasts at the late stage of differentiation. Intriguingly, the CpG motif within iSN40 was not required for its osteogenetic activity, indicating that iSN40 functions in a TLR9-independent manner. These data demonstrate that iSN40 serves as a novel osteogenetic ODN (osteoDN) that promotes osteoblast differentiation. iSN40 provides a potential seed of the nucleic acid drug that activating osteoblasts for osteoporosis therapy.