The ability to detect a broad range of conditions and modifications in human, animal, plant and pathogen genes is highly important in the field of medicine for diagnosing diseases and starting therapy as quickly as possible. Special laboratory equipment is currently required for such tests, which cannot therefore be used in private homes or remote locations. Chemists at the University of Konstanz have now developed a genetic test that does not need to be carried out in the laboratory and can be evaluated with the naked eye, similar to a pregnancy test. The test would therefore be suitable for rapid and simple on-site tests.
A broad range of different and extremely powerful methods is now available for analysing DNA and RNA sequences. These methods are used to identify the base sequence directly or indirectly and most involve the amplification of the nucleic acid region under investigation using a method known as polymerase chain reaction (PCR). Laboratories that carry out PCR-based analyses need expertise, along with specific devices and chemicals. Analyses of this kind require nucleotides, the building blocks used for forming new nucleic acid strands, and polymerases. Such enzymes are found in all living cells; they make exact copies of DNA and RNA, and synthesise new DNA/RNA strands. They are among the most important and sensitive enzymes in PCR analyses.
At the Department of Cellular and Organic Chemistry at the University of Konstanz, Prof. Dr. Andreas Marx and his team of researchers have been experimenting with nucleic acid copying machines known as polymerases for a long time. One of the characteristics of this class of enzymes is their ability to select the right nucleotide for the growing nucleotide chain, thereby ensuring high copying selectivity. Nevertheless, DNA polymerases are also known to use nucleotides with small modifications as substrates, which plays a key role in many biotechnology applications, including the specific labelling of molecules. The chemists from Konstanz have successfully shown that even high-fidelity DNA polymerases are able to use nucleotide chimera modified with horseradish peroxidase as substrates for synthesising a new DNA strand from an existing template, despite the protein being over 100 times larger than the enzyme’s natural substrates. Better still, the enzyme can be used to provoke a colour reaction at single nucleotide resolution that is visible to the naked eye.
“We have been working with polymerases for many years,” says Marx. “We use standard polymerases, but are trying to create nucleotide chimeras with different types of molecules attached in order to synthesise a new DNA strand. We have also successfully increased the size of the attached molecules.” The researchers successfully developed a method involving a polymerase that uses nucleotides coupled to chemically modified horseradish peroxidase to synthesise new nucleic acid strands.1 Marx explains that the error-free linkage of the modified nucleotides depends only on the distance between the molecules, and so the researchers use a connecting piece (linker) to ensure that this distance is always the same. The researchers from Konstanz have filed a patent for their discovery.
The synthesis of the new strand with modified nucleotides follows the same rules as synthesis with unmodified nucleotides. The DNA strand is formed by a polymerase enzyme that reads the DNA template and assembles the new, complementary strand, nucleotide for nucleotide. The researchers from Konstanz link one of the four different nucleotides of the nucleotide mix used to assemble a new strand to a peroxidase molecule. Primers complementary to the sought-after sequences are immobilised on a slide, and nucleic acid sample and reaction mix are added. If the sought-after sequence is detected in the DNA sample, i.e. binds to the primer with the matching sequence, the nucleotide sequence can be synthesised by the polymerase enzyme using normal nucleotides and chimeras consisting of nucleotide and horseradish peroxidase. The presence of the chimeras, and hence the sought-after DNA sequence, can be visualised by adding a colourless dye solution which the horseradish peroxidase enzyme turns reddish-brown within a minute.
Nucleotide chimeras are not only used to detect short sequence stretches, but also point mutations. For this purpose, the researchers design primers that end directly 5' of the mutation site, so that a single modified nucleotide can be inserted. The nucleic acid sequence under investigation can bind, but modified nucleotides can only be added when the sequence contains the sought-after base at this particular site. Moreover, the researchers also have a polymerase enzyme with which they can detect specific viral RNA. The method is therefore suitable for identifying pathogens.
The polymerases used by Marx and his colleagues are so stable that they even work at room temperature, which is quite exceptional. Molecular biology often uses temperature-sensitive enzymes that have to be stored below zero and need temperatures of up 50 °C in order to be re-activated. “We have found a way to freeze-dry polymerases without any loss of activity. We now want to immobilise them on filter paper,” said Marx, going on to explain, “in this form, they are very durable and robust, and the reaction can take place directly on the filter paper. The results are visible to the naked eye. I am sure that this kind of test could work in a way similar to pregnancy tests.” Laboratories would then no longer need expensive laboratory equipment such as thermocyclers, and the analysis could basically be carried out anywhere. A spectrometer would only be needed for exact quantification of the nucleic acids.
“Sensitivity is the only issue we still have to deal with,” says Marx. “Ideally, the test would work with a few drops of blood. But this is not yet possible, and I'm pretty sure that it will be a long time before it is.” The researchers from Konstanz are therefore working on further optimisation of the enzymes and the colour reaction. In addition, over the next few weeks, Marx and his team will spend time writing grant proposals to secure funding for developing the method into concrete applications. “We need a lot of support to produce a prototype,” comments Marx. The researchers are also working on a rapid test for identifying Ebola viruses. “At present in current Ebola areas, blood samples are taken and often need to be transported to laboratories equipped with PCR systems, something that is not always easy in deserts and other remote areas. Rapid tests would enable patients to be tested at home.” The same applies to other pathogens such as noroviruses and influenza viruses. The chemists would also like to offer food tests for detecting pork and horse meat in beef, or finding out whether fruit trees are infested with pathogens in good time before the fruit is harvested. Marx would like to optimise the tests to the extent that everybody could use them. “Patients would be able to carry out a test that they have received from their doctor at home. This is our vision.”
1 Welter M, Verga D, Marx A. Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases. Angew Chem Int Ed Engl. 2016 Jul 8. DOI: 10.1002/anie.201604641