First scissors were invented around 1500 BC in ancient Egypt and for more than 3000 years they have accompanied us us in our daily life. In molecular biology, specialised enzyme “scissors” called nucleases are used for cutting nucleic acids – DNA or RNA.
Molecular “scissors” can be classified based on what and where they cut. Firstly, nucleases can cleave single or double stranded nucleic acids. Secondly, they can be distinguished based on the cleavage site: they cut either at the end of the nucleic acids or within it. These properties make nucleases a crucial tool in molecular biology. In the last 50 years, nucleases which cleave DNA in sequence specific manner facilitated quick progress in molecular biology and their discoverers were awarded a Nobel Prize in Physiology or Medicine in 1978.
In the last decade the scientific interests shifted from DNA to RNA. RNA, like DNA, can store genetic information, but interestingly it also plays many different roles within living cells. This feature made RNA research highly desirable. However thus far, there is no known sequence specific nuclease that could cleave RNA which means that we are not able to easily engineer RNA sequences. That makes working with RNA laborious and expensive.
In our work we studied a nuclease able to cleave double stranded RNA (dsRNA) within its sequence, called Mini-III. To investigate the specificity of this enzyme we combined experimental and theoretical methods. We developed a method for high-throughput sequencing of the ends generated by the “scissors’” cleavage of the RNA. Thanks to that, we were able to check millions of RNA’s ends cut by the Mini-III nuclease. We looked whether this nuclease recognizes a specific sequence within RNA or is sequence independent. Analysis of the sites cleaved by this enzyme helped us to identify a preferred target sequence. It means that Mini-III is the first sequence specific nuclease which cleaves dsRNA.
We wanted to define the precise motif of the RNA sequence which is recognized and cleaved by Mini-III. To do this we created short RNA molecules containing the preferable recognition site for Mini-III and mutated it to see if changes within this sequence influence “scissors” activity. We discovered the motif Mini-III cuts. What’s more, our work identified the distinctive structural element of the Mini- III nuclease responsible and crucial for specific cleavage. This finding is essential for changing the specificity of Mini-III in the future and producing other enzymes specific to different sequences.
Sequence-specific nucleases are a good prototype tool for molecular biology applications. Developing these “scissors” can facilitate work with RNA molecules and studies on RNA structure and function. Moreover enzymes like Mini-III can be used for the detection and characterization of specific RNA molecules in medical tests. They may be used in research for gene silencing or potentially in medical therapy. Range of applications of these molecular scissors is really wide, however still much needs to be done to enable us to use Mini scissors power to the maximum.
About the Author
Dawid Głów is a PhD candidate in the Laboratory of Bioinformatics and Protein Engineering at International Institute of Molecular and Cell Biology in Warsaw. Dawid’s research is focused on sequence specific endonucleases which cleave double stranded RNA. Dawid is also involved in several PhD students’ projects. He co-established ‘Do Science!’, an informal student organization which organizes scientific events like symposiums, discussion panels, seminars and everything that can make science more enjoyable. When he is not doing scientific things, he can be found cooking in his kitchen or playing sports