a) Structures of psoralen-modified uridine. Psoralen with different linkers (S or L) was conjugated to uridine, nine nucleotides from the 5′ end of miRNAs. b) RNA conjugation with psoralen. Gel electrophoresis of deprotected, single-stranded unmodified and Pso-modified miRNAs. c) In vivo RNA photocrosslinking. After transfection with miRNAs, MEF cells were exposed to long wave UV radiation (360 nm) to induce crosslinking followed by UV radiation (254 nm) to photoreverse the Pso-crosslinked product. RNAs pulled down by streptavidin beads were radiolabled with ³²P phosphate and separated by denaturing gel electrophoresis. A crosslinked RNA duplex (XL RNA) with slower electophoretic mobility than single-stranded RNA (SS RNA) is shown.

Enrichment analysis of pulldown RNA from cells transfected with Pso-modified miRNAs: a nontargeting control (miCtrl), miRNA-29a, or miRNA-135b. A total of 13 genes were randomly selected from a list of predicted miRNA-29a and miRNA-135b targets in TargetScan and analyzed by RT-qPCR. GAPDH RNA levels served as an internal control. The average of three independent experiments is expressed as the mean±SEM (standard error of the mean), relative to control.

a) Psoralen-modified miRNA is functional. miRNA-29a (unmodified or modified with psoralen) was transfected into cells together with a plasmid expressing firefly luciferase containing a perfectly matched miRNA-29a target site in the 3′UTR. Firefly/Renilla luciferase (FL/RL) ratios are normalized to those of control miRNAs. b) The biotin signal from Pso-miRNA crosslinked to mRNA is significantly decreased following Ago2 knockdown. MEFs were transfected with transfection agent alone (mock), a control siRNA (siCtrl), siAgo2, or siDicer. After 24 h, all cells were transfected with miRNA-29a (5-S-Pso). Two days later cells were exposed to UVA for 5 min, and total cellular mRNA was isolated using oligo(dT) beads. 30 ng polyA RNA was blotted onto a membrane, and the biotin content of the RNA was detected using a BrightStar BioDetection kit (Ambion).