Fluorescent labeling of RNase H 5′ cleavage products and analyses. (A) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE (B) or capillary electrophoresis (C) after enrichment. (B) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold (left panel) or fluorescent signal (right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. (C) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. (D) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.
