Ribose-seq identifies and locates ribonucleotides in genomic DNA

IMAGE:Georgia Tech Associate Professor Francesca Storici (left), Graduate Student Kyung Duk Koh and collaborators have developed and tested a technique for identifying ribonucleotides in genomic DNA. view more

Credit: Credit: Rob Felt

Ribonucleotides, units of RNA, can become embedded in genomic DNA during processes such as DNA replication and repair, affecting the stability of the genome by contributing to DNA fragility and mutability. Scientists have known about the presence of ribonucleotides in DNA, but until now had not been able to determine exactly what they are and where they are located in the DNA sequences.

Now, researchers have developed and tested a new technique known as ribose-seq that allows them to determine the full profile of ribonucleotides embedded in genomic DNA. Using ribose-seq, they have found widespread but not random incorporation and "hotspots" where the RNA insertions accumulate in the nuclear and mitochondrial DNA of a commonly-studied species of budding yeast. Ribose-seq could be used to locate ribonucleotides in the DNA of a wide range of other organisms, including that of humans.

"Ribonucleotides are the most abundant non-standard nucleotides that can be found in DNA, but until now there has not been a system to determine where they are located in the DNA, or to identify specifically which type they are," said Francesca Storici, an associate professor in the School of Biology at the Georgia Institute of Technology. "Because they change the way that DNA works, in both its structure and function, it is important to know their identity and their sites of genomic incorporation."

A description of the ribose-seq method and what it discovered in the DNA of the budding yeast species Saccharomyces cerevisiae will be reported on January 26 in the journal Nature Methods. The findings resulted from collaboration between researchers in Storici's laboratory at Georgia Tech - with graduate students Kyung Duk Koh and Sathya Balachander - and at the University of Colorado Anschutz Medical School with assistant professor Jay Hesselberth.

The research was supported by the National Science Foundation, the Georgia Research Alliance, the American Cancer Society, the Damon Runyon Cancer Research Foundation, and the University of Colorado Golfers Against Cancer.

Because of the extra hydroxyl (OH) group in the ribonucleotides, their presence distorts the DNA and creates sensitive sites where reactions with other molecules can take place. Of particular interest are reactions between the OH and alkaline solutions, which can make the DNA more susceptible to cleavage.

Ribose-seq takes advantage of this reaction with the hydroxyl group to launch the process of identifying the genomic spectrum of ribonucleotide incorporation. Researchers first cleave the DNA samples at the ribonucleotides, then take the resulting fragments through a specialized process that concludes with generation of a library of DNA sequences that contain the sites of ribonucleotide incorporation and their upstream sequence. High-throughput sequencing of the library and alignment of sequencing reads to a reference genome identifies the profile of rNMP incorporation events.

"Ribose-seq is specific to directly capturing ribonucleotides embedded in DNA and does not capture RNA primers or Okazaki fragments formed during DNA replication, breaks or abasic sites in DNA," Storici noted.

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Ribose-seq identifies and locates ribonucleotides in genomic DNA