We carried out various experiments in order to determine the formation of our desired DNA nanostructure and to investigate the efficacy of the DNA nanostructure in specifically detecting the target, which is the Huntington’s disease miRNA biomarker, Hsa-miR-34b. We assessed the DNA nanostructure assembly using polyacrylamide gel electrophoresis (PAGE), which allowed us to evaluate the desired complementary binding of the various oligonucleotide strands of the structure, as well as the formation of the pre-tetra 2-dimensional structure and the 3-dimensional DNA nanostructure after detection of specific target. .
Figure 1: Formation of 3-dimensional DNA nanostructure from 2-dimensional DNA nanostructure on detection of specific Huntington’s disease biomarker.
Initially, we designed 4 DNA nanostructures and first carried out preliminary investigations in order to assess which of the structures designed were the most successful and should choose for further improvement and evaluation. We did this through assessing the formation of the DNA nanostructures and evaluating and comparing their effectiveness in target detection.
Figure 2: Four designed structures (from left to right) – chosen nanodevice, two-target nanodevice, prism nanodevice, the alternative tetrahedron.
Assessing Formation of DNA Nanostructures
Polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis were used to visualize the formation of the DNA nanostructures. Individual oligonucleotides and DNA nanostructures (and combinations of interacting oligonucleotides for the alternative tetrahedron) were run in order to allow for band comparison for the determination of formation of the nanostructures.
Figure 3: PAGE gel (8%, 70V) showing bands of individual oligonucleotides (O1-O6) of DNA nanostructure, along with target, 2-dimensional nanostructure without presence of target (pre-tetra) and 3-dimensional nanostructure after detection of target (tetra) .
The PAGE gel for the two-target nanodevice was able to show the formation of the nanodevice by giving a gel band of a higher molecular size than that of the individual nucleotides alone. Hence, we could conclude that the individual oligonucleotides were able to come together due to their complementarity to give a nanostructure after the thermal annealing reaction.
The gel bands of the individual oligonucleotides allow for the estimation and verification of the sizes of the oligonucleotides by comparison with the DNA ladder. The formation of the 3-dimensional structure from the 2-dimensional DNA nanostructure in the presence of the specific target can also be clearly observed from the above gel image as a prominent shift from the gel band in lane 9 to the gel band in lane 10 can be distinctly seen.
Figure 3: PAGE gel (8%, 70V) showing bands of different combinations of oligonucleotides.
From the above gel image, it can be verified that oligonucleotides 2 and 3 do not interact with each other. It can also be seen that oligonucleotides 4 and 5 also do not bind to each other complementarily while oligonucleotides 1, 2 and 3 do interact with each other, resulting in a gel band of a higher size compared to the gel bands of the respective individual oligonucleotides. In this way, we were able to analyse the formation of the desired DNA nanostructure from the individual nucleotides.
Figure 4: Fluorescence assay plate reader (Varioskan Flash 4.00.53) measurements showing the detection of specific target with our DNA nanostructure
Referring to the above graph, it can be seen that the fluorescence measured after the detection of the specific target by our DNA nanostructure is higher than that measured before the detection of the target. This implies that the DNA nanostructure can be used to successfully specifically detect the Huntington’s disease biomarker, Hsa-miR-34b.
Figure 5: Fluorescence assay plate reader (Varioskan Flash 4.00.53) measurements comparing the values obtained by the DNA nanostructure from the HKU iGEM 2017 Team with that from the HKU iGEM 2016 Team.
We further performed fluorescence assays in order to compare the DNA nanostructures designed by our team with that designed by the HKU iGEM 2016 team. From the above graph, we concluded that we were able to largely improve the fluorescence absolute values obtained through the assays. However, it can also be seen that the assays may have involved some errors during the measurement as it is implied by the above graph that while the DNA nanostructure from our team is able to successfully detect our specific target, the DNA nanostructure from the HKU iGEM 2016 team is not able to effectively do so. More experiments may be needed to conclusively rule out this possibility.
Further research is still necessary in order to design our DNA nanostructure to give an improved signal and a lower signal-to-noise ratio on detection of the target. Future work may also focus on the provision of a colorimetric signal instead of a fluorimetric one so as to facilitate an easier and faster measurement and interpretation of result and to allow for a more efficient and effective point-of-care diagnostic test.