Aptamer Binding Confirmation

Targeting to Hodgkin’s Lymphoma cells depends on the ability of the PS1NP aptamer to bind with high affinity and specificity. This means that not only does the aptamer have to adhere to a sufficient number of HL cells, it must also not bind to other cell types.

In this experiment, we used flow cytometry to demonstrate that the PS1NP aptamer and aptamer-nanoclew complexes bind to HL cells with high affinity, while interacting with acute myeloid leukemia cells to a much lesser extent.

Introduction

Aptamers are DNA sequences that bind to specific target molecules with high affinity. They are discovered via the Systematic Evolution of Ligands via Exponential Enrichment (SELEX) method, where random sequences are iteratively tested for binding affinity towards a substrate. 

The PS1NP aptamer was identified in this manner to be highly specific towards Hodgkin’s Lymphoma cell lines [1]. It bound to other types of blood cancer cells weakly or not at all. Therefore it is expected that, when tested for its ability to bind to KMH2 (an HL cell line) and HL60 (an acute myeloid leukemia cell line), it would demonstrate high affinity for the former and none for the latter.

Image 1: Folded conformation and sequence of the PS1NP aptamer

Aim

To prove the PS1NP aptamer binds to HL cells with high affinity and specificity even when conjugated to the nanoclew

Techniques Used

Flow Cytometry

Flow cytometry is a method for analyzing protein expression, identifying cell types in a heterogeneous sample, and evaluating cell size and volume [2]. First, cells are fluorescently labeled for the desired marker(s). Afterwards, the cell suspension is fed into the flow cytometer and passed in front of a laser one cell at a time. The machine measures forward and side scatters to determine the size and granularity of cells respectively, in addition to detecting fluorescence signals.

Methodology

Cell Preparation

KMH2 (Hodgkin’s Lymphoma) and HL60 (acute myeloid leukemia) cells were collected and pelleted by centrifuging at 1500 rpm for 5 minutes. After removing the media, the cells were washed twice with PBS, and live cells were counted using a hemacytometer after combining them with 0.4% Trypan Blue in a 1:1 ratio. Cells were then resuspended in PBS to a concentration of 1x10^6 cells/mL. Following, they were fixed through incubation with 4% formaldehyde in PBS for 15 minutes at room temperature. After washing twice with PBS post-fixation, cells were resuspended in binding buffer (10% FBS in PBS).

Sample Preparation

Nanoclews were prepared according to “Sizing and Characterization” (rolling circle amplification time: 6 hours), and aptamers with the FAM fluorophore conjugated to the 3’ end were annealed to them via the protocol on “Annealing Duplexes to Nanoclews”.

Three samples were prepared for both KMH2 cells, while HL60 cells were subjected to only the first two treatments:

  • No treatment (control)
  • Aptamer only
  • Nanoclew w/ aptamers

Samples were incubated at room temperature for thirty minutes, and then washed twice with PBS. Cells were resuspended in 1 mL of PBS before being run in a flow cytometer.

Flow Cytometry

All samples were run and processed in the Life Science Institute’s ubcFLOW facility by Andy Johnson.

Results

Image 2: Comparison between A) untreated KMH2 cells, B) KHM2 cells treated with aptamer only, C) untreated HL60 cells, D) HL60 cells treated with aptamer only, E) untreated KMH2 cells (same as A), and F) KMH2 cells treated with aptamer-nanoclew complex

After treatment with the aptamer alone, KMH2 (Hodgkin’s Lymphoma) cells yield a narrow peak with a clear shift (comparing graph B to A), demonstrating high-affinity binding. Looking at the HL60 (acute myeloid leukemia) cells, the shift of the peak is less apparent (comparing graph D to C), but the distribution is broad with a right-handed tail, suggesting the presence of nonspecific binding. 

With the aptamer attached to the nanoclew, an even more extreme shift occurs (comparing graph F to E), but it is broader than the peak generated with the aptamer alone. This result is expected as the nanoclew essentially creates aggregates of aptamers, making bound cells appear brighter. 

Discussion/Conclusion

As expected, the data demonstrates the ability of the PS1NP aptamer to bind with high affinity to a Hodgkin’s Lymphoma cell line. With regards to its specificity, the decreased intensity of the shift for HL60 cells suggests less aptamer binding, but further experiments involving primary (non-cancerous) cell cultures and better blockage of nonspecific binding is required before drawing broader conclusions. 

For the nanoclew, the attachment of aptamers to it does not seem to inhibit binding ability. However, the determination of the maximum number of aptamers that can be annealed to a nanoclew and the study of how aptamers versus aptamer-nanoclew complexes interact with receptors on the cell’s surface would be useful in further elucidating binding efficiency.

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