Hybridisation probes are widely used as part of DNA profiling analysis in research laboratories as well as in medical diagnostics and forensic laboratories. Despite all the advances in handling, speed and accuracy, there is still room for improvement, especially when it comes to the highly specific detection of nucleic acids.

DNA testing procedures generally exploit the fact that a single-stranded nucleic acid binds to a complementary sequence of a DNA strand, thereby forming a DNA double strand. The probes are labelled with fluorescent (or radioactive) molecules to visualise hybridisation of the probe to its target sequence. Hybridisation can for example be used to identify potential disease-associated transcripts that might be present in the sample under investigation.

Not all DNA sequences are able to form a stable double helix. This is due to the fact that one of the four primary DNA building blocks, i.e. the nucleobase thymine, only binds weakly to its complementary partner, i.e. adenine. The other two nucleobases, guanine and cytosine, bind relatively strongly to each other through three hydrogen bonds. 

It can safely be assumed that nature has exhaustively optimised the base pairing process. Marco Minuth from Prof. Dr. Clemens Richert’s research group in the Institute of Organic Chemistry at the University of Stuttgart, explains why: “DNA in cells is required to fulfil different purposes than in genetic testing procedures. In the former, DNA does not always have to form stable double helix forms, in fact, some situations even require the DNA strands to separate relatively easily and the strands are therefore only held together by weak hydrogen bonds. The strands can open up and the DNA sequence becomes freely accessible to the transcription machinery. It goes without saying that weak bonds between the nitrogenous bases are in this case highly advantageous.”

The different stability of the paired bases can however lead to problems in genetic testing procedures where, for example, a single DNA chip containing tens of thousands of different single-stranded DNA molecules is incubated with a sample under specific physical and chemical conditions. This means that all single-stranded DNA molecules on the chip are exposed to identical conditions. This can lead to false results in cases when the DNA sequences are either particularly rich or poor in A-T pairs. “DNA strands that contain more A-T pairs dissociate at lower temperatures than strands with more G-C pairs. This is because three hydrogen bonds form between G and C, whereas only two bonds form between A and T. In the extreme case, this might lead to false-negative signals,” Minuth explained.

Prof. Richert’s team therefore started to look for a way to make the A-T pair as stable as the G-C pair. “When I started my work, Professor Richert’s team had already found out that an ethynyl group that was attached to a thymine analogue led to a more stable bond with adenine than a thymine analogue without an ethynyl group,” Minuth explained. However, further investigations showed that the thymine analogue to which the ethynyl residue was attached was not ideal as it prevented the two hydrogen bonds from forming. Minuth then made another attempt and his patience eventually led to the discovery of a nucleoside with a stabilising ethynyl residue that was able to form the two hydrogen bonds required. “I was able to link the thymine analogue with the sugar, functionalise the resulting product and synthesise a molecule which we called “E” as it has an ethynylpyridone structure.”

Minuth has already shown that the incorporation of one or two E bases can increase the stability of the hybrid duplexes formed. The E-A pairs are almost as stable as the G-C pairs. The next step is to exchange all thymine bases with E and see what happens. “I can image that better stacking interactions stabilise the double strands to an even greater extent. Such interactions occur when the aromatic rings of the adjacent bases are in a sandwich conformation,” said Minuth optimistically. In addition, Minuth’s experiments have shown that the stability of RNA-DNA hybrid duplexes increases when E is used instead of thymine. This might also have benefits for RNA analyses in which researchers are looking for human mRNA (messenger RNA) which transports the “production plans” for disease-related proteins from the nucleus to the ribosome where the protein is synthesised. 

DNA analyses can benefit from increased selectivity

However, the improved stability of the base pairing between complementary strands is only one reason for using E. Another is the increased selectivity of the nucleoside. Minuth explains: “E binds to adenine with higher selectivity than thymine; it does not pair at all with cytosine, guanine or thymine. Bases that do not normally form base pairs, now fit even less. Steric effects also play a role in the pairing of the bases.” All this has the potential to increase the reliability of test systems. At present the group of researchers has not yet been in contact with users or test developers, but would be happy for their knowledge to be turned to practical use in test methods and are looking forward to inquiries from potential cooperation partners. 

Further information:

University of Stuttgart
Institute of Organic Chemistry
Marco Minuth
Prof. Dr. Clemens Richert
Pfaffenwaldring 55
70569 Stuttgart
Tel.: +49 (0)711 685-64311
E-mail: lehrstuhl-2@oc.uni-stuttgart.de