Dissertation: Reversible base filling may enable new tools for diagnostics and DNA nanotechnology
In his doctoral thesis, MSc Mark Afari developed new types of nucleic acid backbone structures to which DNA bases or their analogues can be reversibly attached. The method can be used, for example, to identify point mutations.
The human genome consists of two complete sets of three billion DNA letters, or base codes, which contain instructions for building and maintaining our bodies. DNA can be imagined as a string of letters of the alphabet that form meaningful words. If one letter is missing or out of place, the message becomes hard to read.
With so many components, this genetic code is also very fragile, and our DNA is constantly under threat. Everyday factors such as sunlight, smoking, poor diet and even natural chemical reactions within our cells can damage or alter it. The most common type of change is a point mutation, the simple substitution of one DNA base, like C, for another, like A, G, or T.
Each day, the cells in our body collectively accumulate billions of these single-letter changes. While many mutations are harmless, some can interfere with a cell’s function or cause it to behave abnormally. If such a mutation is inherited from a parent or occurs early in development, it can affect many or even all of the body's cells—increasing the risk of genetic diseases. Point mutations can have serious effects even when only one DNA letter is altered. Such mutations can disrupt protein function, cause cellular malfunction, or trigger disease such as sickle cell anemia, Progeria (a rare, premature aging disorder), Huntington’s Disease, Cancer etc.
In his doctoral thesis, MSc Mark Afari developed a new method in which a reactive gap in suitably modified DNA or RNA can be filled and then released again by adjusting the pH.
" The method we have developed, called reversible base filling, enables the testing of various modified structures without having to prepare the entire DNA or RNA strand from scratch. On the other hand, by filling a gap in double-stranded DNA or RNA with a suitable natural base derivative, information can be obtained about which base is located at the gap in the complementary strand,” Afari explains.
In DNA, base pairing is mainly controlled by hydrogen bonding and base stacking. The bases (A, T, C and G) are flat and they sit on top of each other in a neat stack inside the twisted ladder shape of DNA. Hydrogen bonds, on the other hand, enable the bases of one DNA strand to attach to the appropriate bases on the opposite strand. Hydrogen bonding acts like Velcro, meaning that the bonds are just strong enough to hold the two sides of DNA together while still allowing the DNA to open when it needs to copy or read genetic information.
The study also examined how quickly bases can be insterted into the gap, identified the key factors that regulate the selection of bases that bind to the gap, and evaluated the stability of our final product after base incorporation.
The study employed the method of dynamic combinatorial chemistry (DCC), a technique that relies on reversible chemical reactions among a pool of compatible molecules under thermodynamic control. In this system, the most stable and favorable products are created naturally and enriched in the product mixture. The researchers synthesised a series of modified oligonucleotides, in which the gap was filled in a reaction with aldehydes to form either an N-methoxy-1,3- oxazinane and N-methoxy-1,3-oxazolidine ring.
The results of the study show that reactions were fast and reversible under mildly acidic conditions (pH 5.5), but much slower at physiological pH (around 7.4), similar to conditions in the human body. This dynamic reversibility acts like a molecular key, allowing the “opening” or “closing” of specific sites in the DNA strand as needed.
The developed method could enable the design of smarter and more flexible tools, for advanced diagnostics or DNA nanotechnology, for example.
Dissertation defense 15 August 2025
Msc Mark Nana Kwame Afari defends the dissertation in chemistry titled “Reversible base filling of oligonucleotide through formation of N-methoxy-1,3-Oxazinane and N-methoxy-1,3-oxazolidine nucleosides” at the University of Turku on 15 August 2025 at 12.00 (Tauno Nurmela Auditorium, University Main Building, Turku).
Opponent: Professor Juan José Díaz Mochón (University of Granada)
Custos: Professor Tuomas Lönnberg (University of Turku)
Contact details: afari.mark@yahoo.com +35846 630 6989
