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The degradation of small RNAs in plants and animals is associated with small RNA 3′ truncation and 3′ uridylation and thus relies on exonucleases and nucleotidyl transferases. ARGONAUTE (AGO) proteins associate with small RNAs in vivo and are essential for not only the activities but also the stability of small RNAs. AGO1 is the microRNA (miRNA) effector in Arabidopsis, and its closest homolog, AGO10, maintains stem cell homeostasis in meristems by sequestration of miR165/6, a conserved miRNA acting through AGO1. Here, we show that SMALL RNA DEGRADING NUCLEASES (SDNs) initiate miRNA degradation by acting on AGO1-bound miRNAs to cause their 3′ truncation, and the truncated species are uridylated and degraded. We report that AGO10 reduces miR165/6 accumulation by enhancing its degradation by SDN1 and SDN2 in vivo. In vitro, AGO10-bound miR165/6 is more susceptible to SDN1-mediated 3′ truncation than AGO1-bound miR165/6. Thus, AGO10 promotes the degradation of miR165/6, which is contrary to the stabilizing effect of AGO1. Our work identifies a class of exonucleases responsible for miRNA 3′ truncation in vivo and uncovers a mechanism of specificity determination in miRNA turnover. This work, together with previous studies on AGO10, suggests that spatially regulated miRNA degradation underlies stem cell maintenance in plants. [ABSTRACT FROM AUTHOR]

We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p 53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL −1 (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL −1 and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation. [ABSTRACT FROM AUTHOR]

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6–99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test. [ABSTRACT FROM AUTHOR]