Inhibitory Impact of 3′-Terminal 2′-O-Methylated Small Silencing RNA on Target-Primed Polymerization and Unbiased Amplified Quantification of the RNA in Arabidopsis thaliana

3′-terminal 2′-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3′ → 5′ degradation and 3′-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3′-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in Arabidopsis thaliana, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3′-terminal 2′-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample.