Sizing and Characterization
Since the size of the nanoclew largely determines the size and shape of the GI-Nc, we must ensure that they are small enough to be endocytosed by cells and assemble into the expected compacted spheres under our reaction conditions.
In this set of experiments, we first synthesized nanoclews via rolling circle amplification (RCA) for a period of 6 or 12 hours. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were then used to determine the size and morphology of the nanoclews.
Endocytosis is a size-dependent process, and clathrin-mediated endocytosis, the process by which the GI-Nc is internalized, has an upper size limit of 200 nm . The size of the GI-Nc is predominantly determined by the nanoclew, which expands in relation to how long for which RCA proceeds. According to Lv et al., a minimum of six hours of incubation is required before compact spherical structures begin to appear . Their assessment of nanoclew growth rate using SEM is shown in Image 1.
Image 1: SEM images of structures present in solution after a certain period of RCA [NATURE PAPER]
However, different papers report different sizes of nanoclews after the same incubation period. While Lv et al. found that the minimum size of nanoclews was 150 nm after 6 hours of incubation, Ruan et al.’s work yielded nanoclews of 68 nm after 12 hours of incubation . Therefore, we predict that structure size is dependent on the template’s sequence, and sought to evaluate our nanoclew using AFM and SEM after 6 and 12 hours of RCA.
To determine the size, structure, and morphology of our nanoclews
Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM) generates images by firing a beam of high-energy electrons at solid samples. The processing of signals stemming from electron-sample interactions yields information about the morphology, structure, and orientation of materials . To improve conductivity, samples are often coated in a thin layer of carbon or metal (in our case, it was gold).
Atomic Force Microscopy (AFM)
Atomic force microscopy (AFM) is a type of scanning probe microscopy. Images are created through the measurement of forces exerted by the sample upon the tip of the probe, thus allowing for the resolution of the three-dimensional topology of the sample’s surface .
The circularized template-primer complex was combined with dNTPs (10 mM) (NEB), 10x BSA (NEB), phi29 DNA Polymerase Buffer (NEB), and phi29 DNA Polymerase (NEB) at the following final concentrations:
|Template-primer complex||300 nM|
|10x phi29 DNA Polymerase Buffer||1x|
|phi29 DNA Polymerase||1000 U/mL|
The sample was incubated at 30C for 6h (AFM) or 12h (SEM). We chose to incubate the sample for longer for SEM because we wanted to use a bigger molecule to clearly elucidate the morphology. Afterwards, the sample was heated to 75C for 10 minutes to deactivate the polymerase,
The samples were then washed twice by centrifugation at 12,000 rpm for 5 min and resuspension in ultrapure water. The nanoclews were finally resuspended in a volume of ultrapure water equal to the original reaction volume.
Atomic Force Microscopy
Nanoclews were first diluted with ultrapure water by adding 1.5x the volume of the current solution. They were then pipetted onto a glass microscope slide (VWR) and left to dry overnight. The machine used was the Nanosurf easyScan 2 system, which was set to operate in an air environment with 200mV free vibration amplitude and auto frequency. The area scanned was 2 x 2 uM, at a rate of 1 s/line and 256 points/line.
Scanning Electron Microscopy
Silicon wafers were cleaned by soaking overnight in aqua regia, washing twice with 100% ethanol, and rinsing with acetone. Ten microliters of undiluted nanoclew solution was pipetted onto the wafers and left to dry at 70C for 2 hours. The samples were then sputter-coated with 5 nM of gold before imaging on the Hitachi S4700 SEM by Derrick Horne at the UBC Bioimaging Facility.
Atomic Force Microscopy
Image 2: AFM images of nanoclews after 6 h of RCA, [WHAT ARE THESE VIEWS CALLED AGAIN NICOOOLLLLLEEEEE]. The arrows point to a single nanoclew beside a large aggregate
Image 2 displays both single and clustered nanoclews, which appear as white circular dots or amorphously shaped clumps respectively. Judging from the image, it can be ascertained that nanoclews are approximate 70 nm in size after 6 hours of RCA, and assembled as expected.
Scanning Electron Microscopy
Image 3: SEM images of nanoclews after 12 h of RCA
To further characterize the morphology of the nanoclew, SEM was performed on nanoclews after 12 hours of RCA. Image 3 demonstrates that nanoclews are composed of interlocked petal-like structures arising from ssDNA folding. It also indicates that after an additional 6 hours of RCA, the nanoclew grew to almost a micrometre in size.
Based on the AFM data, 6 hours of RCA is sufficient for generating properly formed nanoclews small enough to be uptaken via clathrin-coated endocytosis. Additionally, the SEM images showcased the rapid growth rate and internal matrices of the structure. This information suggests that nanoclew size is dependent on both template sequence and RCA time since 6 hours of incubation yields a structure half the size of Lv et al’s, but 12 hours of incubation results in nanoclews almost 20 times the size of Ruan et al’s. However, further time-course experiments are necessary to precisely determine the rate of growth. As a result of our findings, all future experiments involving nanoclews will use structures that have undergone 6 hours of RCA.
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