Figure 1. Ac₄ManNAz, a glycan reporter, incorporates into mammalian cellular RNA.

(A) Schematic of RNA extraction protocol. Ac₄ManNAz, peracetylated N-azidoacetylmannosamine; Prot.K, proteinase K; DBCO, dibenzocyclooctyne.

(B) RNA blotting of RNA from HeLa cells treated with 100 μM Ac₄ManNAz for the indicated amount of time. After RNA purification, Ac₄ManNAz was conjugated to DBCO-biotin, visualized with streptavidin-IR800 (Strep), and imaged on an infrared scanner. Before RNA transfer to the membrane, total RNA was stained and imaged with SYBR Gold (Sybr) to interrogate quality and loading. All subsequent blots were prepared in this manner, and Ac₄ManNAz is always used at 100 μM. The regions where glycoRNAs are present (red text) and non-specific labeling (*) is noted.

(C) RNA Blot of Ac₄ManNAz-labeled HeLa RNA treated in vitro with Turbo DNase or RNase cocktail (A/T1) +/− SUPERaseIn (RNase inhibitor).

(D) RNA Blot of murine RNA after in vivo Ac₄ManNAz delivery via intraperitoneal injection on indicated days at 300 mg Ac₄ManNAz/kg/day. RNA from the liver and spleen were analyzed. Mock (m) mice were injected with DMSO only. RNase treatment was performed on extracted RNA.

See also Figure S1.