Author: Renzo Fenati
Fenati, Renzo, 2019 Optimisation of static and dynamic DNA conditions for biosensing and antimicrobial applications, Flinders University, College of Science and Engineering
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DNA is fast becoming a valuable tool for nanotechnology with its unmatched specificity, and the numerous structures that can be formed. This thesis will look at the interaction of static and dynamic DNA with organic molecules such as intercalators and co-factors.
Static DNA is structured DNA that does not change shape once self-assembled, while dynamic DNA can change conformation, or shape when performing a reaction. By understanding how these two types of DNA interact with other molecules, this will allow for the development of more sensitive biosensors and new applications for DNA nanotechnology. Chapter 1 provides a literature overview of DNA and its various structures and Chapter 2 describes the experimental techniques used in the thesis.
Chapter 3 investigates ways to control the rate and specificity of toehold-mediated strand displacement reactions. These reactions are classified as dynamic DNA processes whereby one single-stranded DNA (ssDNA) displacing sequence (DS) is able to nucleate with a target DNA sequence with a “toehold” double-stranded DNA (dsDNA) that contains two hybridised ssDNA of differing lengths. The DS nucleates at the toehold and then begins a random walk process that displaces the shorter incumbent strand from the target DNA sequence base-by-base, forming hybridised dsDNA and releasing the incumbent DNA strand as ssDNA. This process is also known as three-way branch migration due to the involvement of only three ssDNA strands. The displacement reaction is measured by the decrease in fluorescence of a fluorophore on the target as it duplexes with the DS containing a quencher.
This technique has been found to be useful for genotyping of single nucleotide polymorphisms (SNPs) in short dsDNA sequences (<20 bp). Current three-way branch migration toehold-mediated strand displacement techniques are unable to discriminate between hybridised dsDNA that is perfectly matched (PM) or contains a single mismatch (SM) of base pairs (bp) between the two ssDNA strands when the target DNA sequence is 80 bp long. This can cause issues as it may not be applicable to for genotyping of real-life DNA. Therefore, Chapter 3 investigates increasing the specificity of the toehold-mediated strand displacement process with DNA strands of 80 bp of length. Two fluorophores, ATTO 550 and ATTO 647N,
were investigated for compatibility with the polymerase chain reaction (PCR) process as well as the toehold-mediated strand displacement reaction. The fluorophore ATTO 550 was found to produce non-specific PCR products and so was not used. The addition of ethidium bromide, as well as using the fluorophore ATTO 647N, on the target DNA containing a toehold resulted in an improvement in both the specificity and the rate of the toehold-mediated strand displacement when using DNA strands of 80 bp of length, especially for SMs that involved cytosine. Thus, making the technique applicable to genotyping using real-life DNA.
In Chapter 4, the peroxidase-mimicking activity of different DNA sequences capable of forming static DNA structures, known as G-quadruplexes (G4), that complex hemin (G4/hemin complexes) were assayed. The highest peroxidase mimicking activity was then determined. A G4 is a made up of 1-4 strands of guanine-rich (G-rich) ssDNA sequences that form by stacking guanine-tetrads (G-tetrads) through stabilisation by a mono-cation. The peroxidase-mimicking activity of G4/hemin complexes can catalyse the oxidation of 3,3’,5,5’- tetramethylbenzidine (TMB) in the presence of H2O2 to produce a coloured product and therefore has potential to be used in a biosensor, which is discussed in Chapter 5. Enhancing the rate of the peroxidase-mimicking activity of the G4/hemin complex can allow for more sensitive biosensors. Here, a genetic algorithm (GA) was utilised to screen DNA sequences that form G4/hemin complexes to provide an enhanced rate of peroxidase-mimicking activity. The GA produced 10 new DNA sequences that form G4 from two initial parent DNA sequences. The rate of the peroxidase-mimicking activity of the DNA sequences identified by the GA were then experimentally evaluated using the TMB assay. The DNA sequences that form G4/hemin complexes with an increased rate of peroxidase-mimicking activity were then used in the next round of GA to produce 10 new DNA sequences that form G4/hemin. This was repeated seven times until the rates of peroxidase-mimicking activity appeared to decline, resulting in a DNA sequence with G4/hemin complex showing a rate increase of approximately 5-fold of peroxidase-mimicking activity as compared to the original parent sequences.
At the same time as the screening of the G4/hemin complexes, in Chapter 5 the peroxidase-mimicking activity of surfaces modified with DNA containing G4/hemin complexes were investigated as a potential method to distinguish between hybridised perfectly matched (PM) DNA and SM DNA. This was accomplished by immobilising the DNA sequence G4(G) 001T that formed a G4/hemin complex onto NHS-functionalised Corning R DNA-Bind 96-well microplates. After complexing the G4-containing DNA sequences in the wells with hemin, theG4-containing DNA sequences on the surfaces of the wells were hybridised with PM DNA or SM DNA sequences in separate wells. The wells were then digested with the enzyme T7 endonuclease I, which cuts hybridised SM DNA, but not PM DNA, and releases the G4/hemin
complex from the well. Comparing the peroxidase-mimicking activity of the digested and undigested wells was used to distinguish if the hybridised DNA in the well was PM DNA or SM DNA, and therefore if the DNA sequence added to the well was PM or contained a SM (relative to the DNA sequence attached to the surface of the well). However, it was found that there was no measurable difference between the PM and SM hybridised DNA. Similar experiments were performed using magnetic silica iron core beads, modified with the same G4-containing DNA sequence used in the wells, however, there was also no measurable difference in the peroxidase-mimicking activity of modified beads hybridised with either PM or SM hybridised DNA. This was thought to be due to steric hindrance preventing T7 endonuclease I from approaching the DNA on the surfaces, and therefore not being able to cut the hybridised SM DNA.
Finally, Chapter 6 investigates using G4/hemin complexes as a possible antimicrobial to combat biofilms. Biofilms are made up of bacteria that are protected by extracellular polymer substances, that make conventional antimicrobial treatments against planktonic bacteria (not encapsulated in a biofilm) ineffective, such as antibiotics or peroxide-based (e.g., 3% H2O2 solution) treatments. This can lead to the bacteria becoming resistant to the antimicrobial treatments over time. Here, a modified DNA sequence containing a G4/hemin complex (identified to have the fastest peroxidase-mimicking activity in Chapter 4) was coupled with an antibiotic and tested to determine if the peroxidase-mimicking activity was able to eradicate bacteria inside a biofilm. The G4 was functionalised with an amine on the 5' terminal end of
the DNA sequence (NH2-G4) and was also coupled to the antibiotics fluoroquinolonic acid (FQ) or oxacillin (OX), referred to as FQG4 and OXG4, respectively. However, only OXG4 was able to be purified in concentrations high enough for use in antimicrobial treatments. It was determined using a TMB assay and minimum inhibitory concentration (MIC) assay that the OXG4 retained both high peroxidase-mimicking activity and antimicrobial effectiveness on planktonic Staphylococcus aureus (S. aureus). For the treatment of biofilms, it was found that the OXG4 was more effective than free oxacillin but did not completely eradicate all the bacteria in the biofilm (as determined using CFU assay).
Using the CFU assay, the DNA sequence that was determined to have the highest peroxidase-mimicking activity, complexed with hemin to form G4/hemin complex with no modifications, was also incubated with biofilms and then treated with a 0.5% peroxide (v/v) solution and found to be able to decrease the number of bacteria present in the biofilm by 50%.
Keywords: DNA, G-quadruplex, peroxidase-mimicking activity, surface immobilisation, toehold-mediated strand displacement, genetic algorithm, antimicrobial, antibiotic, biofilm, DNA intercalators
Subject: Forensic & Analytical Chemistry thesis
Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Science and Engineering
Supervisor: Professor Claire Lenehan