Introduction

Receptor-mediated endocytosis, the process by which the GI-Nc is taken into the cell, has an upper size limit of 200 nm [1]. The size of the GI-Nc is predominantly determined by the nanoclew, which can be between 60 nm to 4 µm depending on how long RCA is allowed to proceed. According to Lv et al., a minimum of six hours of incubation is required before compact spherical structures begin to appear [2]. 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 (Lv et. al.)

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 [3]. The discrepancies in literature led us to hypothesize that structure size is dependent on the template’s sequence, so we decided to evaluate our nanoclew using AFM and SEM after 6 and 12 hours of RCA.

Aims

• To determine the size of the nanoclew after 6 and 12 hours of incubation
• To visualize the shape and morphology of the nanoclew

Techniques Used

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, shape, and orientation of materials [4]. To improve conductivity, samples are often coated in a thin layer of carbon or metal (in our case, gold).

Atomic Force Microscopy (AFM)

Atomic force microscopy (AFM) is a type of scanning probe microscopy. Images are created through the measurement of pressure exerted by the sample upon the tip of the probe, allowing for the resolution of the three-dimensional topology of the sample’s surface [5].

Methodology

Nanoclew Synthesis

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:

 The sample was incubated at 30°C for 6 hours (AFM) or 12 hours (SEM). AFM was used to determine the size of the nanoclew intended for incorporation into the GI-Nc. The samples were incubated longer for SEM to generate a larger molecule for the clear elucidation of the morphology. Afterwards, the sample was heated to 75°C for 10 minutes to deactivate the polymerase.

The samples were then washed twice by centrifugation at 12,000 rpm for 5 minutes and resuspended in ultrapure water. 

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 µm, 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 70°C 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.

Results

Atomic Force Microscopy

Image 2: AFM images of nanoclews after 6 h of RCA, represented as a color map (left) and a topographic map (right). 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. From the image, it is ascertained that nanoclews are approximately 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.

Discussion

Based on the AFM data, 6 hours of RCA is sufficient for generating properly formed nanoclews small enough to be uptaken via receptor-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 RCA reaction time.  

However, the nanoclews were found to be half the size of Lv et al’s (70 nm compared to 150 nm) after 6 hours of incubation, but almost 20 times the size of Ruan et al’s (1 um compared to 68nm) after 12 hours of incubation. This indicates that the sequence of the template also contributes to the size of the structure. As a result of these findings, all future experiments involving nanoclews will use structures that have undergone 6 hours of RCA.