The results are described by the study's authors as a breakthrough in the emerging field of epitranscriptomics, which is the study of RNA base modifications corresponding to functionally relevant changes to an organism.

Such RNA base modifications, which are implemented through the biochemical process of methylation, have been identified in both mRNA, which codes for proteins, and miRNAs, which are small noncoding RNA molecules that play a regulatory role by suppressing the expression of a gene.

"Our method can be used for comprehensive analysis and detection of methylation sites in the epitranscriptome, which will allow increased understanding of these methylation events and their mechanisms, changing the landscape of RNA biology and ushering in a new era," corresponding author Masateru Taniguchi, PhD, a professor at the Institute of Science and Industrial Research at Osaka University in Japan, said in a statement.

Designing a tool capable of analysis at the single-molecule level

Taniguchi and his colleagues developed a tool they are calling a "single-molecule quantum sequencer." The tool detects specific target methylations by means of their telltale electrical conductance profiles.

The sequencer is based on a nanoscale electronic device called a "nanogap device" that can perform high-speed electrical discrimination between individual oligonucleotides as they are translocated through the device's nanogap electrodes. The nanogap electrodes were constructed from nanofabricated, mechanically controllable break junctions (MCBJs), which are traditionally used for electrical measurements in the field of molecular electronics.

Illustration of the operating principle of single-molecule quantum sequencing to determine base sequences and identify chemically modified base molecules. Image courtesy of Takahito Ohshiro et al.

Because chemical modifications alter the electrical conductance of the bases, the sequencer can identify specific methylation events based on their conductance profiles identified by the nanogap devices, which were used to measure electrical conductance on an absolute scale in units of pS, as well as a "relative G" value normalized to the electrical conductance of guanine.

The researchers focused on two common base modifications in miRNAs, namely N6-methyladenosine (m6A) and 5-methylcytidine (5mC), which involve the addition of a methyl group to an adenosine (A) nucleotide and a cytidine (C) nucleotide, respectively.

Both m6A and 5mC have been associated with cancer cell propagation and suppression but have proved difficult to detect using traditional methods. Detection of m6A is of particular interest because of its utility in pancreatic cancer diagnosis.

The quantum sequencer identified m6A and 5mC in miRNAs extracted from colorectal cancer cells based on their conductance profiles obtained from the nanogap devices. Since the sequencer relies on individual conductance profiles rather than chemical probes or polymerase chain reaction amplifications, it was possible to detect both methylations, marking the first time that simultaneous detection of m6A and 5mC in the same miRNA molecule had been achieved.

The researchers were able to show that the methylation sites and rates identified by the quantum sequencer were comparable to those determined by MALDI coupled to time-of-flight mass spectrometry. Furthermore, when they evaluated the methylation ratios for each C and A site in the sequences and their relationship at the single-molecule level, the ratios were comparable to those calculated using other methods that are only able to detect a single kind of modification at a time.

The quantum sequencer also revealed that the two types of modification were able to influence each other; specifically, the presence of the m6A modification seemed to facilitate 5mC modification.

"The rate of 5mC methylation is generally affected by the activities of methylation and demethylation enzymes, and so our results imply that the activities of these enzymes can be promoted or deactivated by m6A modifications," Takahito Ohshiro, PhD, the study's lead author, said.

The authors believe their new method represents a step forward in the development of single-molecule RNA sequencing.

"Our technique will allow for greater understanding of the epitranscriptome by enabling the sequencing of various RNA base modifications in their RNA context," the authors wrote.

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