In recent years, there has been huge technological progress in the analysis of genetic material: DNA sequencing techniques have been developed along the lines of faster, further, cheaper. State-of-the-art methods make it possible to handle large projects in a relatively short period of time. However, the error rate is still very high, and samples need to be sequenced several times to determine the correct DNA sequence. Along with research teams from Sweden and Brazil, junior professor Dr. Maria Fyta and her team at the Institute for Computational Physics at the University of Stuttgart have carried out quantum-transport calculations which suggested that specific chemical modifications in the nanopores of sequencing devices would make the sequencing process less error-prone and hence more efficient.
The deciphering of the human genome, completed in 2001, involved numerous scientists around the world, took around 13 years and cost over three billion dollars. Nowadays, such a project would produce a result in just a few days, and it would be cheaper. Next-generation sequencing (NGS) methods in particular have greatly expanded the capacity of research institutes and companies, especially smaller institutes and biotech companies, to analyse genes and genomes. However, these high-tech methods still have a very high error rate. Although this can be compensated with several sequencing reads, the high error rate nevertheless reduces efficiency and produces huge amounts of data that need to be analysed in a time-consuming process that involves huge computational effort.
Many researchers around the world are therefore working on ways to reduce the number of errors while increasing the precision of the methods. Junior professor Dr. Maria Fyta and her group of researchers from the Institute for Computational Physics at the University of Stuttgart are part of the cooperative research centre 716 (Dynamic simulation of systems with large particle numbers) where they have been working with Prof. Dr. Ralph Schleicher from Sweden and Prof. Dr. Rodrigo Amorim from Brazil on optimising methods that make gene and genome sequencing more efficient. They use computer simulations to assess the analysis of genetic information in nanopores. “Nanopore sequencing has been under development for around 20 years,” said Fyta. “Nanopores are nanometre-sized pores that are drilled into different materials, including biological nanopores (e.g. transmembrane proteins) as well as solid bodies such as silicon nitride or graphene, in which electrophoresis takes place.”
Nucleotides have different electronic characteristics in nanopores
The analysis of genetic information using nanopores is considered a third-generation sequencing method. Third-generation sequencing methods are high-tech methods that determine the base sequence by detecting individual molecules. Nanopore sequencing makes it possible – at least theoretically – to sequence a complete human genome in one day for a reasonable price. Nanopore sequencing devices have been on the market for quite some time. “A company called Oxford Nanopore Technologies offers sequencers the size of a memory stick that can sequence several thousand base pairs,” said Fyta. “However, the method is still somewhat error-prone and cannot be used in the medical field.”
Just like many other techniques used to determine DNA sequences, nanopore sequencing involves the electrophoretic separation of nucleic acid fragments of different lengths. The DNA to be analysed is placed in a salt solution and an electric voltage is applied to the nanopores. Almost all methods based on nanopore sequencing use gold electrodes. An electric field forms close to the pores when the negatively charged DNA molecules move and are pulled quickly through the pore. The nucleotide sequence is determined from differences between the four different bases. Although the four bases are chemically very similar, they nevertheless differ in their electronic properties. “The method we are using to determine the DNA sequence measures the transverse tunnelling current across gold electrodes,” says the junior professor. “We detect the difference in the electronic structures of the translocating nucleobases. Different nucleobases and their modifications can thus be determined.”
The researchers from Stuttgart have been carrying out experiments like this for a while. In particular, they performed simulation calculations to assess the behaviour of nucleobases in the tunnelling current, and were thus able to determine the different types of DNA nucleotides. “At some stage we wondered whether it would be easier to differentiate the bases from each other if we coated the gold electrodes in the pores with modified diamond structures,” says Fyta. The researchers found that the differences between the nucleotides become more specific in the tunnelling current. “They were easier to distinguish from each other because, in functionalised nanopores, nucleic acids form a hydrogen bond between the nucleotides and the diamond. Each of the four nucleotides does this in a slightly different way,” said Fyta. “The specificity of hydrogen bond formation in the tunnelling current can be easily visualised using these methods. Our calculations showed that the four nucleotides could be effectively distinguished from each other when diamondoid-functionalised electrodes were used.”1
Diamandoids can be produced and specifically modified by chemists. ”We use tiny hydrogen-terminated diamond structures, so-called diamondoids or diamond cages, for the functionalisation of the surface,” says Fyta. “In addition to the hydrogen terminals, the diamondoids can also be equipped with other functional atom groups that are needed for the formation of hydrogen bonds with nucleotides.” The technology itself should therefore work quite well. However, what the researchers’ calculations have not yet taken into account is the effect of the surrounding medium, i.e. water and salt. Fyta explains: “We still have to find out what effect the salt solution will have on the transport of properties across the electrodes. So far, we have only focused on single nucleotides. We still need to find out what happens when more nucleotides are used.”
Rather than using a traditional-style model for their simulations, the computer physicists opted for a quantum mechanical approach in which the complete system consisting of DNA, nanopore and electrodes is taken into account. “We chose this approach because interactions can be predicted more accurately,” said the junior professor. This kind of simulation takes several days on high-performance computers. But the researchers are in the fortunate position that Baden-Württemberg is home to several supercomputers in university institutes with computing time available, as Fyta explains.
Over the next few months, the scientists will be looking at experimental conditions that they have not yet tested as well as at the system as a whole. “We need to look at the system as a whole, because a single nucleobase in a single nanopore is not what you would normally work with in laboratory experiments. We therefore want to test long nucleic acids in bigger systems,” said Fyta. If all the tests are successful, the researchers plan to develop sequencing devices that have nanopores with diamond-functionalised gold electrodes. However, before all this can happen, practical tests will need to be carried out in the laboratory.
1 Sivaraman, Ganesh; Amorim, Rodrigo G.; Scheicher, Ralph H.; Fyta, Maria: Diamondoid-functionalized gold nanogaps as sensors for natural, mutated, and epigenetically modified DNA nucleotides. In: Nanoscale, 2016,8, 10105-10112, DOI: 10.1039/C6NR00500D