Team:Hong Kong HKU/Results

Experiments and Results

In this project, we designed and contructed 2 DNA nanostructures: NDC and NDC-AS, demonstrated their therapeutic effects on MCF-7 breast cancer cell line, and synthesized them by operating BioBricks in E. coli DH5alpha.

All error bars in graphs indicate 95% confidence interval of triplicates in experiments, unless otherwise specified.

DNA Nanostructures

Construction and Action - Native PAGE

To test the feasibility of our designs, chemically synthesized DNA oligos of the sequences generated by Tiamat were used to assemble the NDCs. DNA-21 and DNA-217, which are DNA equivalents of miR21 and miR217 were initially used as the inputs, before using RNA. Native polyacrylamide gel electrophoresis (PAGE) was used to visualize the component strands, the assembled NDCs and strand displacement outputs suspended in phosphate buffered saline (PBS).

NDC

After assembly, component strands were seen to have formed complexes too large to be ran through the PAGE gel (Fig.1). These large complexes close to the loading well were expected to be successfully assembled NDC that could not pass through the gel due to its large 3D structure, with a tetrahedral base of around 16nm per edge. The band of around 20bp seen below NDC were unbound Strand 5, which was designed to be only partially complementary to Strand 1.

To confirm the assembly of NDC from the 5 component strands, PAGE of structures formed by different strand combinations was done (Fig.2). After annealing, component strands formed complexes larger than their individual sizes, proving DNA structure assembly due to base complementarity. Large complexes close to the bottom of wells could only be seen in samples containing Strand 1, 2, 3 and 4, which together form the 3D tetrahedral base of NDC. These results supported the successful formation of a specific 3D DNA structure.

Our NDC was designed to release to an anti-microRNA oligonucleotide upon binding with intracellular miR21 and miR217. As a preliminary test of strand displacement efficiency, we incubated DNA-21 and DNA-217 with NDC in phosphate buffered saline (PBS) at 37C and ran a page (Fig.3). Band intensities were analysed using software ImageJ.
Successful strand displacement is shown here, because when displaced by 2 inputs, a band of the correct output size was produced and the band intensity, correlated with DNA concentration, was higher than when displaced by either 1 of the inputs.

The specificity of strand displacement was tested using two mutant DNA inputs, 21-5’ mut and 21-3’ mut, each carrying a 6-base mutation at its 5’ or 3’ end respectively. As visualized on the PAGE image (Fig. 4), both 21-5’and 21-3’ mut failed to fully displace Stand 5 to produce the expected output, leaving unbound Strand 5 and inputs as bands of around 30bp and 20bp respectively. The smeared output produced by 21-5’ mut could be explained by its interaction with Strand 5 at sequences outside the expected binding region.

After testing the strand displacement efficiency and specificity by DNA equivalents of mi21 and miR217, RNA of the same sequence as miR21 and miR217 were used as the imputs for more realistic testing. After incubation of NDC with the RNA targets in PBS at 37 degrees Celsius for 30 minutes, displacement of Strand 5 out of NDC by the RNA targets were visualized as bands of size between 35 to 50bp on PAGE.(Fig. 5) This was consistent with the results of strand displacement using DNA-21 and DNA-217. We showed that the design of a DNA nano device with an output of RNA-DNA duplex was feasible and our NDC could work in a more realistic simulation.

NDC-AS

After assembly, component strands were seen to have formed complexes too large to be run through the PAGE gel (Fig.6). These large complexes close to the loading well were expected to be successfully assembled NDC that could not pass through the gel due to its large 3D structure, with a tetrahedral base of around 16nm per edge. With extended complimetary region between Strand 1-AS and Strand 5-AS.

Same as how we tested strand displacement of NDC, we tested NDC-AS using RNA-21 and RNA-217. After incubation with the RNA targets in PBS at 37 degrees Celsius for 30 minutes, NDC-AS gave the expected output as in Lane 6. Displacement of Strand 5-AS out of NDC by the RNA targets were visualized as bands of size around 50bp on PAGE.(Fig. 7) This was consistent with the results of NDC, showing that NDC-AS also produced the expected output in the presence of RNA targets, despite carrying an extra aptamer.

NDC-AS Construction - Thioflavin T Fluorescence Measurement

To test if the AS1411 aptamer on NDC-AS was successfully formed, we incubated 1μM of our nanostructures with 10μM Thioflavin T at room temperature for 10 minutes. Thioflavin T is a selective sensor of DNA G-quadruplex. Thioflavin gives off fluorescence upon binding to G-quadruplex due to its restricted rotation and enforced planarization [1]. High fluorescence signal after incubation of NDC-AS with Thioflavin T would indicate successful formation of AS1411, which has a G-quadruplex structure [2]. Thioflavin T was incubated with water only as control.

NDC-AS, after incubation with Thioflavin T, emitted significantly higher fluorescence than NDC without AS1411. This shows that G-quadruplex was formed in NDC-AS.

Loading, Release and Strand Displacement - Fluorescence Measurement

Doxorubicin (Dox) loading and release

Doxorubicin is a self-fluorescent molecule with maximum excitation at 480nm and maximum emission at 595nm [3]. Fluorescence quenching occurs when doxorubicin intercalates into DNA duplexes [4]. To characterize the maximum loading capacity of the Nano Drug Carrier (NDC), we incubated doxorubicin with NDC and measured the fluorescence signal decrease. The point of maximum quenching was an estimate of the ratio of loading of doxorubicin onto NDC, ie. number of doxorubicin molecules on each NDC. When incubating in ratio exceeding the maximum quenching , excess doxorubicin showed low affinity to NDC.

After intercalating into DNA, doxorubicin's intrinsic fluorescence will be quenched. Using the property, we designed fluorescence measurement experiments to determine the drug loading capacity of the NDC. Doxorubicin was incubated with assembled NDC in room temperature for 1 hour at different ratios. The maximum concentration of doxorubicin used was 6 μM such that all the fluorescence signal would be in the linear range.

Dox loading

Figure 9 shows that with higher concentration of NDC to incubate with, the fluorescence signal continues to decrease. There was a linear decrease from 0 to 0.1 μM of NDC. And another linear range which shows the minimum signal, was between 0.3 – 0.5 μM of NDC. The linearity at range 0 to 0.1 μM of NDC added would be explained by the maximum binding of the NDC, in which there are excessive doxorubicin. The linear range at 0.3 – 0.5 μM of NDC would be explained by the complete quenching, which indicated that no free doxorubicin was present in the solvent. The range 0.1 – 0.3 0.3 – 0.5 μM of NDC is not linear. In which there would be a system of equilibrium between free doxorubicin and bound doxorubicin. It would be explained by our mathematical model.

Approach used in Figure 10 started with a fixed NDC concentration, with varying doxorubicin concentrations. Linearity is observed in 0 – 2 μM of doxorubicin, and the slope increases after 2 μM of doxorubicin. A turn is observed in 2 μM of doxorubicin, indicating NDC to doxorubicin ratio more than 1:20 will lead to the decrease the quenching ability of the NDC.

Dox release

The fluorescence signal (Excitation: 480nm, Emission: 595nm) produced by NDC (0.3 μM) loaded with Dox (in 1:20 ratio) in different pH of phosphate buffered saline (PBS). NDC loaded with doxorubicin was incubated at 37 degree Celsius in 384-well plate. PBS was pre-titrated using hydrochloric acid in 37 degree Celsius water bath, such that after the addition of neural samples, the final pH reaches the desired pH at 37 degree Celsius. Plate was prewarmed for 10 minutes to reach 37 degree Celsius before taking the initial reading (0 minute). Readings were taken per 5 minutes for 1 hour.
The unbinding of doxorubicin from DNA double helix results in the de-quenching of doxorubicin fluorescence signal. The increase in fluorescence signal hence proves the release of doxorubicin from NDC.

In the above figures, fluorescence ratios of sample to free dox are plotted against time for the first 1 hour (Fig.11 and Fig.12). As seen from dropping free doxorubicin fluorescence, doxorubicin could be photobleached[5]. This calibrates the effect of photobleaching in a short period of time. However, due to photo recovery of signal of doxorubicin at 16, 24, 48 hour, it is not favorable to calibrate the fluorescence signal to free dox since photo recovery of free dox leads to the underestimation of fluorescence signal.

Fluorescence of drug loaded NDC was divided by fluorescence of 6 μM doxorubicin in the same plate to calibrate the effect of fluorescence photobleaching. As shown, fluorescence signal of NDC in pH 5 increases significantly in the first 20 minutes of incubation whereas others remains relatively consistent. However, the fluorescence signal of drug loaded NDC at pH 5 doesn’t reach that of 6 μM doxorubicin, showing incomplete release, and an equilibrium is reached since the binding and release are reversible. The release of Tb and Td have similar trends with Td having slightly lower release ratio.

Strand displacement

We have shown in the above PAGE formation of desired displacement outputs using inputs DNA-21 and DNA-217. By fluorescence measurement, we hoped to better study the dissociation of Strand 5 and from the nanostructure upon binding to inputs. We assembled NDC and NDC-AS using FAM-conjugated Strand 1 and Iowa Black®-conjugated Strand 5. Displacement of Strand 5 away from Strand 1 could be detected by fluorescence of FAM, freed from the quenching by Iowa Black®.

Much higher fluorescence of FAM was detected in NDC displaced by DNA-21 compared to NDC without input. This confirmed effective displacement of Strand 5 by DNA-21. Displacement by DNA-217 was lower, since it was designed to bind distal to the toehold and did not participate in strand displacement. This result implies that strand displacement was the main reason of increase in fluorescence in this experiment.
NDC-AS displaced by DNA-21 showed lower fluorescence than NDC. This was expected because NDC-AS was designed to have extended complementary region between Strand 1 and Strand 5, to increase displacement specificity. Nonetheless, NDC-AS displaced by DNA-21 also showed significantly higher fluorescence than when displaced by DNA-217 and no input. It can be concluded that NDC-AS also functioned as expected in the presence of target sequences.

Therapeutic Effects

Entry - MCF-7 fluorescence microscopy

Entry of NDC and NDC-AS into MCF-7 cells was analyzed under the fluorescence microscope. DAPI, a fluorescent stain that binds strongly to AT-rich region of DNA, was used to stain the nuclei of MCF-7 in blue. Doxorubicin is a self-fluorescence molecule which has a maximum excitation of 480nm, and maximum emission at 595nm [3]. With the natural red fluorescent signal of doxorubicin, the ratio of number of cells with doxorubicin in red to number of nuclei in blue can be calculated. A high ratio suggests greater accumulation of doxorubicin in the cells.

Though entry of NDC-AS was not found to be significantly higher than NDC, both nanostructures when loaded with doxorubicin, resulted in significantly higher intracellular doxorudicin signal than doxorubicin alone.
Though AS1411 did not facilitated drug entry here, we would like to investigate whether it has extra cancer cell killing effect due to its binding to nudleolin. Therefore, we conducted a cytotoxicity assay.

Action - MCF-7 MTT cytotoxicity assay

To show the therapeutic effect of our NDC and NDC-AS, we tested their ability to kill MCF-7 human breast cancer cell line in MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay. Our MCF-7 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum, 20μg/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured at 37 °C with an atmosphere of 5% CO2.
MTT assay is a colorimetric assay for assessing cell metabolic activity. The sample optical density (OD) is therefore inversely correlated with cell death. Percentage cell death can be calculated with the formula: Cell death(%) = 1- Sample OD/Control OD x 100% . In our assay, Sample OD is the mean OD of drug-treated cells, while Control OD is the mean OD of non-treated cells.
Cells were plated at a density of 1e5 per well in 96-well plates to reach 80% confluence. Doxorubicin (150μM), Doxorubicin-loaded NDC (150μM Doxorubicin + 1μM NDC) and Doxorubicin-loaded NDC-AS (150μM Doxorubicin + 1μM NDC-AS) was diluted 5 times in a serial 10X dilution, before being added to the cells in wells. After 24 hours incubation, 20μl MTT was added into each well and incubated for 4 hours. 100μl DMSO was then added and incubated with shaking for 10 minutes. Finally, the optical density (OD) due to formation of purple formazan crystals was measured by microplate reader at 570nm.

The preliminary cytotoxicity study revealed that both NDC and NDC-AS were able to kill MCF-7 breast cancer cells within this range of dilution. However, free doxorubicin only exhibited cytotoxicity at higher concentrations (ie. when less diluted). This may be explained by increased cell entry of doxorubicin as shown in fluorescence microscopy. In this assay, NDC-AS exhibited overall higher cytotoxicity than NDC. This may be due to extra inhibition of cell growth by the nucleolin aptamer AS1411. In the future, we hope to replicate this assay, to confirm added therapeutic effect of NDC and NDC-AS compared to free doxorubicin.

ETHERNO: E. coli-synthesized Therapeutic Nanostructures

To confer E. coli. the ability to synthesize single-stranded DNA for nanostructure assembly, we cotranformed DH5alpha with 3 plasmids. The first plasmid is pSB1C3 containing our parts. The second plasmid codes for a modified Human Immunodeficiency Virus reverse transcriptase. The third plasmid codes for Murine Leukaemia Virus reverse transcriptase. These two reverse transcriptase plasmids were gifts from Voight Lab, MIT [6]. Each cotransformed bacterium would then be able to synthesize 1 of the 5 component strands of NDC or NDC-AS.

Successfully cotransformed bacterial colonies were picked and inoculated in Terrific Broth for overnight culture. This step is for mass production of the component strands we needed. After that, the single-stranded DNA synthesized inside the bacteria were extracted using TRIzol® Reagent and chloroform. At the phase-separation stage, only the top aqueous phase, which contained RNA and DNA single strands, was collected. The collected nucleic acids were then digested with RNAase A overnight to remove the RNA, leaving behind the DNA componant strands.

To check if these strands actually could form NDC and NDC-AS, we used them to assemble the nanostructures by PCR annealing as described above. The previously conducted PAGE was repeated. These E. coli-synthesized NDC and NDC-AS were also visualized under transmission electron microscope.

PAGE

The collected single-stranded DNA (ssDNA) were suspended in water which contained leftovers of chemicals used in extraction. Because of this, these ssDNA could not be easily visualized on PAGE, as performed above for NDC assembled from chemically synthesized ssDNA.
None the less, we tried to amplify the ssDNA extracted using primers that overlapped with the component ssDNA sequences. We expected to get back bands of the expected ssDNA sizes as shown in Fig.1 and Fig.6 on PAGE. We also annealed the strands synthesized by ETHERNO, to see if large complexes similar to NDC and NDC-AS as shown in Fig.1 and Fig.6 could be produced.

Expected bands of Strand-1, Strand 1-AS, Strand 5 and Strand 5-AS can be seen in Fig.20a, which means these 4 strands were present in the ssDNA extracted from ETHERNO. Other strands could not be amplified here, may be due to problematic primer design. Designing primers for these nanostructure component strands was challenging due to the complicated secondary structures formed.
Since these extracted ssDNA were able to form large structures that could not pass through PAGE.(Fig. 20b), we moved on to look for any successfully assembled NDC and NDC-AS under transmission electron micrpscopy.

Transmission Electron Microscopy

5 μl of NDC and NDC-AS samples was first adsorbed onto glow-discharged 400 mesh copper grids (Ted Pella, Inc.) for 1 minute followed by staining with 2% uranyl acetate for 1 minute. Stained samples were washed twice by distilled water to remove excess uranyl acetate. Stained NDC and NDC-AS samples on the grid were visualized using a Philips CM 100 Transmission Electron Microscope with 100 kV operating voltage.