4 research outputs found
Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing
Enzymatic synthesis
of RNA nanostructures is achieved by isothermal
rolling circle transcription (RCT). Each arm of RNA nanostructures
provides a functional role of Dicer substrate RNA inducing sequence
specific RNA interference (RNAi). Three different RNAi sequences (GFP,
RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures
(Y-RNA). The template and helper DNA strands are designed for the
large-scale in vitro synthesis of RNA strands to prepare self-assembled
Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced
by its physical structure and different gene silencing activity is
achieved depending on its arm length and overhang. In addition, enzymatic
synthesis allows the preparation of various Y-RNA structures using
a single DNA template offering on demand regulation of multiple target
genes
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>
Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes <i>In Vivo</i>
Establishment
of an appropriate cell labeling and tracking method
is essential for the development of cell-based therapeutic strategies.
Here, we are introducing a new method for cell labeling and tracking
by combining metabolic gylcoengineering and bioorthogonal copper-free
Click chemistry. First, chondrocytes were treated with tetraacetylated
N-azidoacetyl-d-mannosamine (Ac<sub>4</sub>ManNAz) to generate
unnatural azide groups (-N<sub>3</sub>) on the surface of the cells.
Subsequently, the unnatural azide groups on the cell surface were
specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged
dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free
Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented
strong NIRF signals with relatively low cytotoxicity and the amounts
of azide groups and DBCO-650 could be easily controlled by feeding
different amounts of Ac<sub>4</sub>ManNAz and DBCO-650 to the cell
culture system. For the <i>in vivo</i> cell tracking, DBCO-650-labeled
chondrocytes (1 × 10<sup>6</sup> cells) seeded on the 3D scaffold
were subcutaneously implanted into mice and the transplanted DBCO-650-labeled
chondrocytes could be effectively tracked in the prolonged time period
of 4 weeks using NIRF imaging technology. Furthermore, this new cell
labeling and tracking technology had minimal effect on cartilage formation <i>in vivo</i>