Abstract
Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.
Highlights
Physical disruption of the cell membrane and DNA transport are of fundamental interest in cell biology, biophysics and soft materials.[1,2] Application of electric pulses to disrupt the cell membrane is a simple, easy and popular technique to deliver nucleic acids such as DNA and RNA into living cells
Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague
The results of this study provide a mechanistic understanding of DNA translocation across an electro-pore which is necessary for understanding DNA translocation across real cell membranes, and for predictable loading and dosage control of nucleic acids into vesicles using electroporation
Summary
Physical disruption of the cell membrane and DNA transport are of fundamental interest in cell biology, biophysics and soft materials.[1,2] Application of electric pulses to disrupt the cell membrane (electroporation) is a simple, easy and popular technique to deliver nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) into living cells. The transport mechanism of these nucleic acids, especially DNA molecules, across the cell membrane during electroporation, is poorly understood.[3,4,5] The cell membrane is a complex entity comprising phospholipids and various lipid domains, and inclusions such as membrane proteins and cholestrol.[6] A dense cytoskeleton network known as actin cortex is present underneath the cell membrane.[7] inferring the mechanism of DNA translocation across the cell membrane by conducting experiments on cells is inherently a complex and a challenging task due to simultaneous involvement of several cell membrane and cytoskeleton entities. An important step towards understanding the transport mechanism of DNA across the cell membrane is to decouple several cell membrane and cytoskeleton entities and obtain rudimentary knowledge about the transport process by using lipid vesicles as cell membrane models.[5]
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