PepMute™ siRNA Transfection Reagent

Description:
PepMute™ siRNA Transfection Reagent is a novel siRNA delivery platform inspired by viral cell-penetrating peptides (CPPs). It enables exceptionally high gene silencing, achieving over 95% knockdown at 1 nM siRNA across a variety of mammalian cell types.

Using our proprietary Peptide Simulation Technology (PST), hundreds of viral CPPs were computationally simulated, synthesized, and screened for gene delivery efficiency in diverse mammalian cells (Figure 1). Through this process, PepMute™ was identified and validated as an exceptionally effective vector for condensing and delivering short (under 100 bp) single- or double-stranded nucleic acids, including siRNAs, miRNA mimics, and DNA oligonucleotides, to a broad spectrum of mammalian cells.

Figure 1. A cartoon showing PepMute™ siRNA Transfection Reagent was developed by PST

Size:
– PepMute™ Reagent, 1.0 mL, sufficient for ~1,333 reactions based on transfecting 0.5~5.0 pmol siRNA or miRNA mimics in a 24-well plate
– PepMute™ Transfection Buffer (5x ), formulated for maximal transfection efficiency, 8.0 mL at 5x concentrated stock solution to make 40 mL of working solution

Applications:
– siRNA, miRNA mimics, or mRNA transfection
– DNA/siRNA co-transfection
– DNA oligos transfection

Storage:
Store at 4 °C for PepMute™ Reagent and RT for PepMute™ Transfection Buffer (5x ). If stored properly, the product is stable for 12 months or longer.

Advantages:
– Excellent silencing at final 1.0 nM siRNA
– Over 95% gene silencing in a wide variety of cells
– One tube reaction with easy standard protocol
– Compatible with serum and antibiotics
– Protocols adapted to HTS
– Very low cytotoxicity
– Very affordable

Silencing Efficacy Comparison between PepMute™ siRNA Transfection Reagent and Leading Products

Figure 2. Excellent silencing of endogenously expressed KIF11 (also known as EG5) in HEK293 cells with 1.0 µl of PepMute™ reagent and 0.5 pmol EG5 siRNA per well of 24-well plate. KIF11 (also known as EG5) encodes a motor protein that belongs to the kinesin-like protein family involved in chromosome positioning and bipolar spindle formation during cell mitosis. A reduction in KIF11 levels causes mitotic arrest. PepMute™ reagent effectively delivers EG5 siRNA (final 1.0 nM) to HEK293 cells, leading to more than 80% of “round-up” phenotype of HEK293 cells 48h post transfection over negative control (final 1.0 nM with sham EG5 siRNA) while leading siRNA transfectin reagents, Lipofectamine™ RNAiMAX (RNAiMAX, 1.0 nM EG5 siRNA) / INTERFERin (1.0 nM EG5 siRNA) / Dharmafect (10.0 nM EG5 siRNA) and jetPRIME (20 nM EG5 siRNA) give average 37%, 23%, 53% and 48% ball-shaped phenotype respectively on HEK293 cells. The phenotype of “rounded-up” 293 cells was visualized (upper panel) and quantified (lower panel) 48h post-transfection with a Nikon microscope.

Figure 3. Silencing efficiency comparison of PepMute™ Transfection Reagent (upper panel) with Dharmafect 4 (middle panel) and Lipofectamine™ RNAiMAX (RNAiMAX, lower panel) siRNA Transfection Reagents on A549 cells. siRNA targeting renilla luciferase at different final concentrations ranging from 0.5 to 20 nM was co-transfected with the renilla luciferase gene (0.5 µg of pRL-CMV DNA per well) by the above three transfection reagents per manufacturer’s protocols into A549 cells growing on a 24-well plate. Renilla luciferase activity was determined 36h after post co-transfection with the renilla luciferase determination system (Promega). The luminescence was measured from 5.0 µl of lysate during 10s integration with a luminometer (Beckman Coulter LD 400). Luciferase activity was expressed as light units integrated over 10s (RLU) and normalized per mg of cell protein by using the BCA assay. The error bars represent the standard deviation derived from triplicate experiments. Luciferase-silencing efficiency was calculated relative to untreated cells. While PepMute™ and Dharmafect™ 4 reagents delivered significant gene silencing from 1.0 nM of renilla luciferase siRNA, Lipofectamine™ RNAiMAX gave good knockdown only after 20 nM, while enhanced gene expression at low concentrations of siRNA (0.5 and 1.0 nM, respectively) was observed.

Figure 4. PepMute™ Transfection Reagent knocked down stable GFP expression in MCF7 cells (upper panel) and U2OS cells (lower panel) by reversely transfecting 5.0 and 1.0 nM GFP siRNA, respectively. Green fluorescence protein (GFP) was stably expressed in MCF7 and U2OS cells. siRNA targeting GFP gene (right panel) and a sham siRNA (left panel) were introduced into MCF7 and U2OS cells with final 5.0 and 1.0 nM, respectively, by reverse transfection with PepMute™ Transfection Reagent. GFP gene silencing was monitored 48h post-transfection by a Nikon fluorescence microscope. Quantitative analysis showed that GFP siRNA at 5.0 and 1.0 nM delivered by PepMute™ siRNA Transfection Reagent knocked down 90% and 95% stably expressed GFP in MCF7 and U2OS cells, respectively.

Data Sheet & Protocol
– A Protocol for siRNA Transfection and DNA/siRNA Co-Transfection
– A Short Protocol for siRNA Transfection for Advanced Users 
– A Short Protocol for siRNA/DNA Co-transfection for Advanced Users 
– A Protocol for DNA Transfection to Mammalian Cells