Development of ratiometric fluorescent pH nanoprobe to study endocytic uptake pathways of antisense oligonucleotides

• Main Author: Liew, Donn Hou Feng
• Other Authors: Phan Anh Tuan
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Science::Biological sciences::Biophysics; Science::Physics::Optics and light
• Online Access: https://hdl.handle.net/10356/148512
• Institution: Nanyang Technological University

Anti-sense oligonucleotides (ASOs) are single short strands of synthetic DNA/RNA designed to complement specific RNA sequences. Antisense therapy as a treatment option has received widespread consideration as only knowledge of a nucleotide sequence is necessary for drug design. However, one challenge of antisense therapy concerns attaining effective delivery to intracellular targets. Furthermore, the endocytic pathways taken by ASOs in different conditions are poorly understood. In this thesis, we have developed a ratiometric fluorescent pH nanoprobe to track antisense oligonucleotides (ASOs) as these ASOs negotiate different endocytic pathways in U2OS cells. The number of ASO-containing endosomes were observed to decrease across time indicating the possibility of degradation in the lysosomes or recycled to the cellular plasma membrane. However, the rate of degradation was observed to depend on ASO concentration. Therefore, usage of our developed ratiometric fluorescent pH probe was paramount for futher investigation. We observe a greater distinction between pH profiles of endosomal population for U2OS cells incubated with a higher concentration of ASO. Our observations suggest that cellular incubation with a higher concentration of ASO results in cellular uptake of ASOs via endocytic pathways not conventionally utilised by ASOs at a lower concentration of incubation.