1.

Student Name:

2. Dissertation Title: Quantification of DNA Modifications using DNA-Gold Affinity Interactions

3. Summary of Project:

More recently a new method for detecting DNA methylation has been developed ( Chem. Commun., 2014, 50,
13153). The method relies on the base dependent affinity interaction of DNA with gold. The aim of this project
is to establish essential design and operational criteria for utilising this phenomenon in detecting other DNA
modifications. Measuring DNA modification is of great interest to biology, however such information is
notoriously difficult to access via conventional methodologies as samples typically require cumbersome
chemical treatment, amplification and readout. By exploring the underpinning science of this interaction, we
will develop a new platform technology for detecting chemical modifications of DNA.

4. Rationale - brief review of relevant research in the field:

The nature of the affinity interaction (i.e., adsorption) of DNA molecules on gold surface has long been
regarded as ‘‘non-specific’’ and ‘‘difficult to control’’. Recent advances in this field, pioneered by the Tarlov and
Mirkin groups,1,2 have suggested that this is a base dependent phenomenon, and follows conventional
physisorption and chemisorption mechanisms. Because this adsorption is highly base dependent, 1 it can be
used to distinguish two sequentially different DNA molecules. However, it was not until 2014 when our group
demonstrated a method referred to as “Methylsorb” or “eMethylsorb”,3-5 that the use of the gold-DNA
adsorption process gained real momentum in distinguishing two DNA sequences with different base
compositions. This work gave a completely new dimension to the field not previously considered by any other
groups. It also allows us to focus our research to study the effect of other chemical changes of a “ native” DNA
molecule on the nature of the adsorption process, and to uncover new fundamental scientific principles and
challenges of this process in a biological context.

These chemical changes could occur via chemical modifications of

native DNA molecules - a process where the functional groups
(e.g., methyl, hydroxymethyl, etc.) are added onto a specific base
of the DNA sequence - mainly on cytosine.6,7 These chemical
modifications do not change the DNA sequence, but alter the way
genetic information is read by the cell’s protein machinery, and
plays a critical role in cell function. 6 These additional groups pose
unique fundamental issues and challenges which could directly
affect the nature of the adsorption process and magnitude of the
gold-DNA affinity strength. Such an example is the methylation of
cytosines in genomic DNA. The additional methyl groups could
themselves interact with the gold surface (via hyperconjugation) 8
or alter the intrinsic ability of cytosines to interact with gold by
altering the nanomechanical properties of DNA molecules (e.g.,
flexibility and molecular dynamics). 9 As these additional groups
could significantly alter the magnitude of the gold-DNA
adsorption strength, the accurate measurement of this Figure 1. Proposed method for the
differential adsorption affinity strength could be useful in detection of chemical modifications of
understanding the biological functions of chemically modified DNA molecules using gold-DNA affinity
bases in the DNA sequences. For example, aberrant patterns and interactions.
levels of modified DNA bases across a cell’s genome play a
significant role in modulating gene function and specific cellular pathways during progression of many diseases
including cancer and neurodegenerative diseases. However, to date no one has attempted to use the
differential adsorption affinity of chemically modified and unmodified DNA molecules for DNA modification
detection in the context of biological applications.

The chemical modifications of DNA can be detected by simply monitoring the adsorbed DNA molecules onto a
bare gold electrode. The adsorption process can also be monitored in real-time with surface Plasmon
resonance (SPR) detection (See Figure 1). We can overcome almost all the current hurdles involved in the
conventional detection methodologies for DNA modification detections. Some of these are illustrated in Figure
1.
References:
(1) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J., J. Am. Chem. Soc. 2003, 125, 9014-9015
(2) Storhoff, J. J.; Elghanian , R.; Mirkin , C. A.; Letsinger, R. L. , Langmuir 2002, 18, 6666–6670
(3) Sina, A. A. I.; Carrascosa, L. G.; Palanisamy, R.; Rauf, S.; Shiddiky, M. J. A.; Trau, M., Anal. Chem. 2014, 86, 10179-85.
(4) Sina, A. A. I.; Howell, S.; Carrascosa, L. G.; Rauf S.; Shiddiky, M. J. A.; Trau, M., ChemCommun. 2014,50, 13153-6.
(5) Koo, K.M.; Sina, A. A. I.; Carrascosa, L. G.; Shiddiky, M. J. A.; Trau, M., Analyst2014, 139, 6178-84
(6) Jones, P. A.; Takai, D., Science 2001, 293, 1068-1070
(7) Plongthongkum, N.; Diep, D. H.; Zhang, K., Nat. Rev. Genet. 2014, 15, 647–661
(8) Denis, A.; Pullman, A., Theor. Chim. Acta1967, 7, 110-116
(9) Severin, P. M. D.; Zou, X.; Gaub, H. E.; Schulten, K., Nucleic Acids Res. 2011, 39, 8740–8751.

5. Significance of the study:

It is now widely recognised that the chemical modifications of DNA is one of the key components of gene
expression in cells and has been shown to be involved in the progression of many diseases including cancer
and neurodegenerative diseases. Development of a platform method for the direct detection of chemical
modifications in unprocessed DNA samples would transform the detection of these diseases. This project aims
to develop a platform technology for the highly efficient detection of chemical modifications of DNA that
avoids the need of conventional bisulfite treatments, amplification or associated surface modifications steps.
The world market for such a technology is substantial. The epigenomics market was valued at $4.1 billion in
2012, up from $263.2 million in 2006, and is growing rapidly. It is highly likely that advances made by the
research in this program can capitalise on these markets through the generation of Australia-based intellectual
property and the development of appropriate commercialisation prospects.

6. Research plan, methods and technologies:

Theresearchplanisdividedintofivemainsteps,whichwillbeconductedinastep-wise manner:
(i)design,fabricationand characterization ofdevices/chips (SPR,electrochemistry)
fordetectionofmethylation/hydroxymethylation/acetylationusingmodelsyntheticDNAsamples,(ii) Extraction of
theDNAsamples from cancer celllines,(iii)pre-treatment,processingforthe detection,(iv)directadsorptionof the
sampleongoldandthedetectionoftargetsbySPRorelectrochemistry,and(v)development
ofamultiplexedandpointofcare devicewiththecapabilityofanalysingmultipletargetsforregional analysis
withCpGresolution. To this end an especial focus will be made for the development of a multiplexed DNA
methylation device. This will be done by coupling the developed method with existing multiplex-PCR
technology. Multiplex-PCR allows amplifying multiple genomic regions in one step. By further isolating each
region of interest within the PCR pool using magnetic-pull down method, the individual regions will be delivered
onto the electrodes printed on a multiplexed chip for specific analysis of their adsorption levels.

Devices/chipswillbemadeonsiliconsubstrate bystandard
photolithographytechniquesfollowedbygoldcoatingofthesensing
area.TheDNAsamplesderivedfromcelllineswillbedirectlyadsorbedonthegoldchips/
electrodesanddetectedinrealtimeand label-freemanner.Operation
ofthedeviceisextremelysimple,sinceadsorption ofDNAinducesmasschanges atthesensingarea
whichtriggeropticalorelectrochemicalchangesonthebiosensingdevice.
7. Timeline:
HDR milestone Project Aims
Confirmation (1)Design,fabrication,and characterization ofthedevices/chips;
(1st year) (2)Optimization of the operational parameters.

Mid-candidature Perform key experiments and theoretical analysis to explore the

(2nd year) fundamental mechanisms underlying interactions between gold and
chemically modified DNA molecules.

Thesis Review and 1. Developmentofapoint-of-caredevice(multiplexed)forclinicalapplications.

Submission(3rd year) 2. Thesiswritingandreview.

8. Specialist Equipment and Resources:

Theprojectrequiresasolidknowledgeinvariousanalyticaltechniques(SPR,electrochemistry, etc), and surface

materialchemistry. Knowledge
andexperimentalskillindesign,fabricationandcharacterizationdevices,materialscharacterization,
andelectrochemicaland optical measurementsareespeciallyimportant.

Requiredfacilitiesincludethefabricationandcharacterization ofmicrofluidicdevices(QMF
facility),materialcharacterizationequipments(SEM,TEM,XRD,FTIR,NMR,etc.),readout systems(home-
madeSPRsystem, electrochemicalworkstation,etc.), and biological samples.

Ifopportunitiesarise,fundingtoattendlocaloroverseasconferencestoaidinmyresearch wouldbehighlybeneficial.