DNA sequencing provides insight into the blueprint of living cells and individuals and is now used in many aspects of biological sciences, medicine, biotechnology and forensic sciences. An ultrafast way to sequence DNA is to read the nucleotide letters of single DNA strands as they pass through a protein nanopore. A new study by the team of Prof. Han Remaut (VIB-VUB Center for Structural Biology) in collaboration with Oxford Nanopore Technologies presents a nanopore that is engineered to hold two constrictions. Such dual constriction nanopores are shown to be advantageous in resolving regions of DNA more difficult to read by conventional nanopores and support the generation of very high accuracy nanopore sequencing data. Their work appears in Nature Biotechnology. 

DNA sequencing in modern medicine
The sequence of our DNA holds a treasure in information. It is a molecular identity card that comprises the information on the working of our cells.

Sequencing parts or the entirety of our DNA (our genome) can inform us about the risk for inherited diseases, or the presence and progression of cancer, for example. Also, sequencing the DNA or RNA present in our saliva or stool can identify the presence of infectious disease agents such as bacteria and viruses, and can predict how these will respond to antibiotic therapy. Not surprisingly, DNA sequencing is taking an increasingly important role in modern medicine and large efforts are made to make reading our DNA fast and cost efficient.  

In 2014, the UK company Oxford Nanopore Technologies released a powerful new way to sequence DNA: nanopore sequencing, first used on a pocket-sized device called the MinION. In this technique DNA strands are passed through a protein nanopore (a nanometer sized channel) that resides in an electrical field. The passage of the different nucleotides (DNA ‘letters’) alters the electrical current that is generated by ions flowing through the nanopore. 
In this way, DNA can be ‘read’ at hundreds of letters per second in small high-tech devices that fit in the palm of your hand – or larger ones that can sequence many whole human genomes at a time. Some regions of DNA can be more difficult to read by the available nanopores, however. This is particularly true for homopolymer regions, stretches of DNA where the same letter is repeated. 

Current sequencing flow cells (“R9” series) offered by Oxford Nanopore use a nanopore that is a modified version of CsgG, a protein borrowed from bacteria. Bacteria use CsgG channels to transport other proteins to the cell surface. Through extensive engineering the CsgG channel was reshaped into a nanopore with optimal properties for DNA sequencing. The lab of Han Remaut studies the biological role of CsgG channels in bacteria. 
First author of the study Sander Van der Verren says: “Using electron cryo-microscopy I studied how CsgG cooperates with a partner protein called CsgF and found that CsgF binds partially inside the CsgG channel to make a second constriction or narrow point.” 

A 2-constriction reader
In nanopore sequencing the pore constriction has a dominant contribution to the electrical signals that allow the reading of passing molecules such as DNA. The researchers reasoned that having two consecutive constrictions may be advantageous to sequence hard-to-read regions of DNA. 
In a joint effort with the nanopore team at Oxford Nanopore, they showed that a fragment of CsgF can also introduce a dual constriction in the engineered CsgG nanopores used for DNA sequencing. When using these prototype dual constriction nanopores to sequence DNA, the teams found that having two constrictions made it easier to decipher the number of letters in homopolymer regions of the DNA. 

A version of the dual constriction nanopore which is included in the R10 flow cells provided by Oxford Nanopore, has shown promising results in resolving homopolymer regions, supporting the generation of consensus accuracy of 99.999% on small genomes, and in addition high single molecule accuracy nanopore data[1].  These promising results open the road to further engineer this biological pore complex into a tool that helps read DNA with the highest possible accuracy, using minimal amounts of material and time. 

Han Remaut, PhD, said: “At VIB, we’re constantly on the lookout to see how studying fundamental biological processes can inspire new ways to build powerful technological tools and help solve medical and societal needs.”

Oxford Nanopore Technologies does not disclose the precise design of the nanopores that are used in their devices; over time they have engineered several thousands of designs of nanopores, many of which have broad structural similarities but intricate differences which can optimise performance.
Flow cells containing two different types of pores are currently available: R9.4.1 and R10.3;  ONT has spoken about the fact that R10 uses a dual constriction concept, along with specific analysis methods, to support improved analysis of particular regions, for example longer homopolymer repeats. For the most up to date performance information on both pores you can visit www.nanoporetech.com/accuracy

Sander E. Van der Verren, Nani Van Gerven, Wim Jonckheere, Richard Hambley, Pratik Singh, John Kilgour, Michael Jordan, E. Jayne Wallace, Lakmal Jayasinghe and Han Remaut (2020). A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity. Nature Biotechnology. Doi: 10.1038/s41587-020-0570-8

FWO Flanders through a PhD aspirant fellowship to Sander Van der Verren. EC Horizon 2020 through ERC consolidator grant  ‘BAS-SBBT’, grant agreement no. 649082

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