Phosphorylatable short peptide conjugation for facilitating transfection efficacy of CS/DNA complex.
Previously, we had demonstrated that enhancing intracellular unpacking of exogene from its chitosan carrier by promoting chitosan degradation could markedly improve transfection efficiency of the CS/DNA complex. In this article we addressed a novel strategy of phosphorylatable short peptide modification for further facilitating intracellular DNA unpacking and optimizing transfection efficiency of the CS/DNA complex. A short peptide (SP) with the amino acid composition of "LLLRRRDNEY*FY*VRRLL" containing two potentially phosphorylatable tyrosine residues was synthesized. The short peptide could be phosphorylated by constitutively expressed cytoplasmic protein kinase Jak2. The SP was conjugated to chitosan and combined with GFP/luciferase reporter gene plasmid DNA to form SP-CS/DNA complex. In vitro phosphorylation and DNA releasing assays verified that mammalian cell lysate could effectively phosphorylate SP and hence promote plasmid DNA unpacking from the SP-CS carrier. Thereafter, C2C12 myoblast cells were transfected by SP-CS/DNA and the transfection efficiency was presented by the expression of GFP and luciferase reporters. Further more, multiple cell lines were transfected by SP-CS/DNA complexes loading luciferase reporter gene. Results revealed that, compared with CS, SP-CS could intensively augment the transfection efficiency to the level of near lipofectamine 2000.
Sun
Bei
B
Tianjin Medical University, Institute of Endocrinology, Tianjin 300070, China.
Zhao
Ronglan
R
Kong
Fanqiang
F
Ren
Yuping
Y
Zuo
Aijun
A
Liang
Dongchun
D
Zhang
Jingyu
J
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008055:Lipids; D008156:Luciferases / Q000235:genetics; D051379:Mice; D053758:Nanoparticles; D010316:Particle Size; D010766:Phosphorylation; D010957:Plasmids; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan; D008156:Luciferases