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The University of Southampton
Chemistry

Research project: Stulz: Chemistry-Biology-Medicine interface

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We use modified nucleotides and ODNs in biology and medicine to target a number of issues of high societal importance; in particular gene expression and diagnostics profit highly from the use of synthetic DNA. We are pursuing several systems to address regulation of gene expression, targeted cell killing, sensing of ODN markers and photodynamic therapy; the majority of the applications are targeted at cancer therapy.

We have developed DNA based membrane nanopores, which form stable inclusion complexes in lipid bilayers. Together with the groups of Stefan Howorka (UCL) and Ulrich Keyser (Cambridge) we have studied several types of pore forming DNA systems ( HOT paper in Angew. Chem. 2013, Nano Lett. 2013). We found that two porphyrins are sufficient to form very stable insertions for a six-helical DNA bundle. The simple design allows for the creation of a pore of approximately 5 nm width and 14 nm height, with an inner pore diameter of 2 nm. Current recordings of the ionic flow across the lipid bilayer confirms the formation of the nanopore.

In addition, we could show that a 6-porphyrin containing DNA duplex inserts into the lipid bilayer and induces a flow of ions along the phosphate backbone. This leads to the formation of the smallest possible DNA nanopore – a single DNA duplex (Nano Lett. 2016). It also reveals that it will not be possible to completely suppress ion flows in larger DNA nanopores, e.g. by blocking the channel.

We are now investigating the nanopores in targeted cell killing.

 

Figure 1: Modelled structures of the DNA origami nanopore made of 6 DNA helices (left), and the smallest possible porphyrin-DNA nanopore (right).
Figure 1:

 

Figure 1: Modelled structures of the DNA origami nanopore made of 6 DNA helices (left), and the smallest possible porphyrin-DNA nanopore (right).

 

 

Porphyrins have shown to be perfectly well suited to create electrochemical sensors for DNA. Together with the group of Jerzy Radecki and Hanna Radecka in Olsztyn, Poland, we are using cobalt metallated porphyrins that show to be ultrasensitive towards hybridisation with the complementary sequence (Chem. Commun. 2014). The sensor is very selective, able to discriminate single nucleotide polymorphism, and extremely sensitive: with the use of gold nanoparticles on the surface of the electrode, our sensors have a detection limit in the attomolar range and can sense as few as 23 DNA molecules (Chem. Commun. 2018).

 

Figure 2: A highly sensitive genosensore based on porphyrin-modified DNA probes and gold nanoparticle coated electrodes
Figure 2:

 

 

Figure 2: A highly sensitive genosensore based on porphyrin-modified DNA probes and gold nanoparticle coated electrodes.

 

 

 

 

A novel approach which could lead to a simple forensic analysis of short tandem repeat (STR) sequences is being explored using self-assembled fluorescent nucleosides. We used porphyrazines which showed a large increase in fluorescence when assembled on complementary di- or tri-nucleotide repeats (EurJOC 2018). With this, we could discriminate several forensically relevant loci and directly determine the number of STRs, which is informative on the allelle. This would overall give the same readout as with forensic DNA analysis which to date is done using PCR. We are now working on using different fluorophores to distinguish more relevant genomic loci. This research was highlighted in EurJOC in the section Outstanding Organics: Functional Organic Materials, as well as in a press release from Diamond Light Source: New forensic DNA profiling technique on the horizon. 

Figure 3: Self-assembled porphyrazine-nucleosides on DNA for direct readout of forensically relevant STR numbers.
Figure 3

 

Figure 3: Self-assembled porphyrazine-nucleosides on DNA for direct readout of forensically relevant STR numbers.

 

 

 

Funding Provider:

Institute for Life Sciences

https://www.southampton.ac.uk/ifls/index.page

CRUK

https://www.cancerresearchuk.org/

EPSRC

https://epsrc.ukri.org/

Related research groups

Chemical Biology, Diagnostics and Therapeutics
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