Figure 5: Digestion of ^m5C-modified duplex oligos (GCWGC site) by BisI or NhoI.

The 5′-FAM-labeled top strand contains two ^m5C bases (G^m5CAG^m5C) and the bottom strands contain either two (G^m5CTG^m5C), one (G^m5CTGC, internal C methylated), or no ^m5C bases (GCTGC), respectively. Thus, the annealed oligos contain a total of four, three, or two ^m5C. P1 (20 bp) and P2 (14 bp) are the cleavage products. P3 is a possible top-strand nicked intermediate (NI) due to asymmetric nicking of the top strand. The substrate (sub, 34 bp), P1, and P3 were detected by FAM fluorescence imaging. (A,B). BisI- and NhoI-digested duplex oligos (four, three, or two ^m5C) analyzed on a 15% PAG-urea denaturing gel. C. Partial digestion of the duplex oligos by NhoI and the DNA products were analyzed on a 20% TBE (non-denaturing) gel, stained by SYBR Gold and imaged by fluorescence imaging. The 5-bp dsDNA size marker (Fermentas) and the single-stranded oligos (IDT, 20–100 nt) were used to estimate the size of the cleavage products.