N6-Methylation Assessment in Escherichia coli 23S rRNA Utilizing a Bulge Loop in an RNA-DNA Hybrid.
Kyoko YoshiokaRyoji KuritaPublished in: Analytical chemistry (2018)
We propose a sequence-selective assay of N6-methyl-adenosine (m6A) in RNA without PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA-DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by an anti-m6A antibody with a high affinity. By employing a surface-plasmon-resonance measurement or using a microtiter-plate immunoassay method, a specific m6A in the Escherichia coli 23S rRNA sequence could be detected at the nanomolar level when synthesized and purified oligo-RNA fragments were used for measurement. We have successfully achieved the first selective detection of m6A2030 specifically in 23S rRNA from real samples of E. coli total RNA by using our immunochemical approach.
Keyphrases
- escherichia coli
- nucleic acid
- single molecule
- transcription factor
- high throughput
- circulating tumor
- cell free
- dna methylation
- label free
- genome wide
- gene expression
- pseudomonas aeruginosa
- staphylococcus aureus
- real time pcr
- klebsiella pneumoniae
- loop mediated isothermal amplification
- multidrug resistant
- living cells
- fluorescent probe