title: Sequence-specific nuclease-mediated release of cells tethered by oligonucleotide phospholipids.
abstract: Single-stranded oligonucleotide-conjugated lipids (ssDNA-PEG-lipids) that associate with the cell membrane confer to the cell an artificial adhesive capability via sequence-specific hybridization to complementary oligonucleotides, forming bonds of double stranded oligonucleotides (dsDNA). Such artificial tethers permit surface patterning of cells or controlled formation of cellular aggregates. However, the hybridization responsible for tethering cells to surfaces or to other cells is not trivially reversed under physiological conditions. In this study, we approach the unbinding of tethered cells by cleaving dsDNA bonds with restriction endonuclease BamHI or digesting bonds with the nonspecific nuclease Benzonase. The procedure was applied to CCRF-CEM cells bearing dsDNA suspended in isolation, cells tethered to glass substrates, and cells aggregated heterotypically with other ssDNA-bearing cells. Cells liberated from surfaces with BamHI could be flushed from flow chambers and viably recovered while the majority of cells not bearing enzyme recognition sequences were retained on the surface, and DNA-tethered cells could be nonspecifically recovered viably from surfaces after Benzonase treatment. Heterotypic aggregates of cells joined by recognition sequence DNA could be dispersed with 10 min exposure to BamHI while undispersed cells heterotypically aggregated with a control sequence remained. Likewise, 10 min exposure to Benzonase was sufficient to disperse aggregates independently of sequence. The potential to undo artificially engineered DNA-mediated adhesion offers new possibilities in the controlled arrangement of cells relative to other cells and in the study of membrane biophysics.
authors.affiliation: Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-Ku, Kyoto, Japan.
authors.forename: Ian T
authors.initials: IT
authors.lastname: Hoffecker
authors.affiliation: Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-Ku, Kyoto, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Takemoto
authors.affiliation: Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-Ku, Kyoto, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Arima
authors.affiliation: Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-Ku, Kyoto, Japan. Electronic address: iwata@frontier.kyoto-u.ac.jp.
authors.forename: Hiroo
authors.initials: H
authors.lastname: Iwata
mesh_terms: D002460:Cell Line; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D010743:Phospholipids; D004720:Endonucleases
keywords: Cell patterning; Cell–cell adhesion; Membrane modification; Multicellular aggregate; Restriction enzyme; ssDNA