Please use this identifier to cite or link to this item: https://doi.org/10.1038/srep37721
Title: GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells
Authors: Fazil, M.H.U.T
Ong, S.T
Chalasani, M.L.S
Low, J.H
Kizhakeyil, A
Mamidi, A
Lim, C.F.H
Wright, G.D
Lakshminarayanan, R 
Kelleher, D
Verma, N.K
Keywords: antisense oligonucleotide
cytoskeleton protein
locked nucleic acid
oligonucleotide
sorting nexin
stathmin
cell culture
gene silencing
genetic transfection
genetics
human
physiology
pinocytosis
procedures
T lymphocyte
transport at the cellular level
Biological Transport
Cells, Cultured
Cytoskeletal Proteins
Gene Silencing
Humans
Oligonucleotides
Oligonucleotides, Antisense
Pinocytosis
Sorting Nexins
Stathmin
T-Lymphocytes
Transfection
Issue Date: 2016
Citation: Fazil, M.H.U.T, Ong, S.T, Chalasani, M.L.S, Low, J.H, Kizhakeyil, A, Mamidi, A, Lim, C.F.H, Wright, G.D, Lakshminarayanan, R, Kelleher, D, Verma, N.K (2016). GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells. Scientific Reports 6 : 37721. ScholarBank@NUS Repository. https://doi.org/10.1038/srep37721
Rights: Attribution 4.0 International
Abstract: Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCÎ?, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics. © 2016 The Author(s).
Source Title: Scientific Reports
URI: https://scholarbank.nus.edu.sg/handle/10635/178753
ISSN: 20452322
DOI: 10.1038/srep37721
Rights: Attribution 4.0 International
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