Fig 1.
Removal of rRNA from total RNA.
A principle of poly (A) enrichment. B rRNA depletion using magnetic pulldowns. C RNase H-based rRNA removal. D artificial ribosomal RNA remover (ARRR). E Cleavage mechanism of benzimidazole catalysts shown in 1D.
Fig 2.
The artificial nuclease (ARRR) cleaves 28 S rRNA into two fragments.
A Using ARRR, the DNA-catalyst conjugate 1v splits the 28 S rRNA into fragments of approximately 4,000 and 1,200 nucleotides, in contrast to the unconjugated probe 2v. RNase H digestion yields identical fragments with both probes. RNAs were separated on an 2% Agarose-TAE Gel. Stained with GelRed. B DNA probe 1v and 2v target sequence and cleaving site on the human 28 S rRNA.
Fig 3.
Optimization of reaction conditions.
A total RNA was cleaved by ARRR with pH values ranging from 7–8.6 and then separated using a BioAnalyzer pico assay. B 2% Agarose gel of ribosomal RNA treated with various amounts of cleaver mix and cleaved by RNase H and ARRR. C 2% Agarose gel of RNA mixed with 20 eq. cleaver mix as described before, but the temperature was adjusted to 37°C or 50°C for all experiments.
Fig 4.
Targeted cleavage of 28 S rRNA reveals that ARRR has little off-target effect.
A Total RNA was treated with an all rRNAs cleaving probe mix (28 + 18), a 28 S specific probe mix (28) or the 28 S specific mix where potential 18S targeting probes were excluded (28 v.2). RNAs were separated on an 2% Agarose-TAE gel and stained with GelRed. B The same experiment as in A was loaded on two Agilent BioAnalyzer pico chips for denaturing chip gel electrophoresis and merged into one graph.