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Department of Physics

The Cavendish Laboratory
 

RNA is omnipresent, it is found anywhere from the genetic material of SARS-CoV-2 to RNA drugs and mRNA vaccines. However, RNA sequencing is a labour-intensive process and suffers enzyme biases, which leads to the loss of native RNA information including RNA identity and quantity. RNA identity is needed for cancer and disease variants diagnostics, gene expression quantification and RNA makeup identification - such RNA modifications can make mRNA vaccine more stable or the structures themselves can act as targets of antiviral drugs.

We focused on DNA origami over decades as a building material. Surprisingly, our study demonstrated that RNA ‘makeup’ can be detected by using similar principles to reshape RNA to linear RNA origami codes.  Professor Ulrich F. Keyser

RNA or ribonucleic acid is one of the key biological molecules that bridges our genetic information (DNA) and molecular factories (proteins). RNA has a single strand in comparison to double-stranded DNA helix. Labour-intensive approach of RNA sequencing suffers enzyme biases that causes the loss of native RNA information including RNA identity and quantity. Up until now, we could not detect RNA ‘makeup’,  including its chemical modifications and overall shape. The mere order of bases in RNA could not tell us how that RNA looked.

Researchers at the Cavendish Laboratory, University of Cambridge have recently developed a new method - Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) that has made possible the identification of multiple RNAs in parallel. They employed their method to analyse native RNA molecules, including ribosomal RNA molecules that can be used as species-specific markers. In their paper ‘Nanopore microscope identifies RNA isoforms with structural colours’ published in Nature Chemistry, they describe how the approach can discriminate between different structural variants of RNA, known as RNA isoforms, relevant to many biomedical applications. Through ARTEMIS, they have identified multiple RNA isoforms of the enolase gene in human total RNA deriving from cervical adenocarcinoma, as well as isoforms of non-coding RNAs, crucial for gene expression regulation. 

The ‘Target RNA’ is translated into RNA origami codes with designed sets of complementary DNA strands. As with Lego bricks the researchers combine fitting units to create a linear RNA:DNA duplex that can bear a code. Each code has multiple sites. Here, instead of only having ‘1’ and ‘0’ bits, they introduced up to ten bits thus enabling production of 10 billion RNA codes (with ten sites). RNA codes are then read with a nanopore microscope. Nanopore microscope works via voltage-driven translocation of negatively charged RNA origami codes through a small orifice towards a positively charged electrode in an electrolyte solution. It translates RNA code into a current signal with ‘super-resolution’. “We live in the RNA era, where diseases, pathogens, therapeutics and even vaccines are based on RNA molecules. Here, we show characterisation of RNA with simple short DNA binding to it. By creating this hybrid duplex from RNA and DNA we bypass problems with degradation and enable understanding of RNA in its native form.” shares Filip Bošković, PhD student from Cambridge’s Cavendish Laboratory. 

ARTEMIS has the potential to create ~1010 unique RNA IDs that are readable using nanopore microscopy or imaging methods. This opens new avenues for single-molecule mapping of RNA motifs important to drug development. In addition, the study also demonstrates that highly abundant natural RNAs may serve as scaffolds for nanoscale self-assembly technique known as DNA origami with a wider length range and yield. This can help in detecting vaccine byproducts with potential side effects in pre-clinical trials. Using RNA-based energy-saving data storage and cryptography instead of DNA data storage can offer new ways of synthesis at the nanoscale. Most diseases are classified by a change of a few transcripts. Thus, accurate identification of RNAs of interest has to bypass prior amplification and reverse transcription biases.

“We focused on DNA origami over decades as a building material. Surprisingly, our study demonstrated that RNA ‘makeup’ can be detected by using similar principles to reshape RNA to linear RNA origami codes.” Professor Ulrich F. Keyser from Cambridge’s Cavendish Laboratory.


Reference: Bošković, F., Keyser, U.F. Nanopore microscope identifies RNA isoforms with structural colours. Nat. Chem. (2022). https://doi.org/10.1038/s41557-022-01037-5

Image by Nic0leta from Pixabay

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