Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores

Biochemical determination of inosine tail lengths. (A) Scheme for analyzing inosine tails based on RNase A resistance of purine homopolymers. (Top) Poly(A)+ mRNAs, (bottom) nonpolyadenylated RNAs. (Left to right) Tailing by Cid1, 3′ end labeling by T4 RNA ligase and ³²P-pCp, RNase A digestion. (B) Cid1 adds ∼50 inosine residues onto the poly(A) tail of a model poly(A)+ mRNA. (Left) Labeled products without (−) and with (+) I-tailing, and without RNase A digestion, run on a 6% denaturing polyacrylamide gel. (Right) Labeled products without (−) and with (+) I-tailing after RNase A digestion, run on a 12% denaturing polyacrylamide gel. Markers are indicated as for Figure 1. (C) Cid1 adds a long heterogeneous tract of inosine residues onto the 3′ end of a model nonpolyadenylated mRNA. Lanes are as for B above. (D) Cid1 adds a long heterogeneous tract of inosine residues onto the 3′ end of the nonpolyadenylated 5.8S rRNA. Lanes are as for B above.