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Researchers at Brown Discover RNA Ligase That Functions at High Temperatures

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NEW YORK (GenomeWeb) – Brown University researchers have discovered an RNA ligase that has the ability to function at high temperatures, potentially enabling detection of RNA mutations and improving RNA sequencing.

In a study published in RNA Biology the researchers note this is only the third RNA ligase from a thermophile to be studied, and the others are single-stranded ligases that are inefficient at strand joining.

The ligase, called KOD1Rnl, comes from a heat-loving archea called Thermococcus kodakarensis that was originally discovered in a sulpherous volcanic steam vent on Kodakara Island in Japan.

The researchers used an "a holistic screening approach" to discover KOD1Rnl, Anubhav Tripathi, a bioengineer at Brown University and principal investigator on the RNA Biology study, told GenomeWeb in an email. The group specifically sought out thermostable RNA ligases that might be efficient in nick joining, so that they could be used in RNA detection and sequencing, Tripathi said. High heat can be used to straighten out RNA molecules, making things like hybridization easier.

Tripathi's lab began looking for the ligase while undertaking a project on rapid detection of Ebola, due to the fact that other available ligases were not up to the task. The ligase was then used to detect Ebola RNA transcripts as well as to detect the presence of point mutations.

"We tested four combinations of mutations on the Ebola transcripts, and the ligase demonstrated exceptional specificity in reporting them," Tripathi said, adding that the specificity varied from 10-2 to 10-4.

According to the study, the addition of single-stranded DNA ligase to the reactions increased template-dependent ligation efficiency. And, importantly, pretreatment with heat-labile proteases overcomes inhibition inherent in human serum samples.

To give a sense of potential applications, a ligase called T4 RNA ligase 2 is similar to KOD1Rnl. According to one recent review, T4 RNA ligases have become essential for a subset of rapid amplification of cDNA ends (RACE) protocols, 3' RNA labeling, and for the preparation of microRNA sequencing libraries.

Tripathi, meanwhile, sees three potential categories of use for KOD1Rnl.

It could be employed in place of RT-PCR or other reverse transcriptase-based methods to detect RNA via RNA-based probe ligation, and could also be used to detect RNA mutations.

The ligase might also be useful for RNA-mediated oligonucleotide annealing, selection, and ligation, or RASL-seq, Tripathi said, particularly because it is thermostable and template dependent. The RASL-seq method has been recently described as applicable to massively parallel quantitative analysis of gene expression.

Finally, KOD1Rnl could be used as a tool to generate elaborate oligonucleotide structures, adapters, or probes, Tripathi said.

A patent is pending for the ligase through Brown University. "We are actively looking for partners in the molecular diagnostics and NGS sample prep fields to bring this to market," Tripathi said.

At least one firm is designing molecular diagnostics around a thermostable enzyme discovered in an extremophile organism. The OmniAmp enzyme used by Lucigen is a polymerase with innate reverse transcriptase activity that was discovered in a virus inhabiting a Yellowstone National Park hot spring.

Lucigen also provides its OmniAmp enzyme in a kit for loop-mediated amplification of RNA and DNA at temperatures up to about 160 degrees Fahrenheit. New England Biosciences makes a similar kit, the WarmStart LAMP Kit for DNA and RNA, that uses a blend of Bst 2.0 DNA polymerase and RTx reverse transcriptase.

In addition, research efforts like the Extreme Microbiome Project and the Earth Microbiome Project will likely add to the database scientists can use in the future to uncover more molecular biology reagents with unique properties.

Tripathi's group is now actively working on simplification and automation of both NGS and PCR-based diagnostics, he said. Characterization of the KOD1Rnl represents the first achievement in "a continued effort to discover and develop more efficient, cost effective, and unique enzymes for NGS and molecular diagnostics," Tripathi added.

He is also part of a collaboration to develop a ligation-on-RNA method that eliminates the need to create a cDNA library from RNA and merges ligation and amplification steps to detect single point mutations, as previously reported, with applications for low-cost HIV point mutation detection.

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