Part:BBa_K3224002

Survivin shRNA-like siRNA

shRNA or small hairpin RNA are artificially designed RNA which has a stem-loop structure. This part is a 25-base containing small interfering RNA (siRNA) synthesized in-vitro, with 3-bp mediated self-complementarity formed between their two termini, introduced to form an shRNA-like structure. This forms a structure of short stem containing 3-bp, and a large loop containing 22-bp, which houses the silencing sequence. The 5’ end of the stem region is conjugated with the fluorophore 6-FAM (which has an excitation wavelength of 495 nm and an emission wavelength of 520nm) and the 3’ end is conjugated with the quencher IB-FQ.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

CHARACTERIZATION

To compare the relative efficiency of gene silencing by Survivin shRNA (BBa_K3224001) and Survivin shRNA-like siRNA (BBa_K3224002) by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) analysis to evaluate and thus compare the effectiveness of cargo delivery by Lipofectamine 2000 and the chimeric CPP BP100-(KH)₉.

cDNA synthesis was carried out by incubating 2.5 μg of RNA with 1 μl of oligo dT primers, 1 μl of dNTPs, 1 μl of reverse transcriptase in 4 μl of RNase transcription buffer, 2.5 μl of RNase inhibitor. 1 μl of cDNA was mixed with the Master Mix. The analysis was carried out according to the manufacturer’s protocol.

qRT-PCR was done to compare the relative gene expression of Survivin using GAPDH as control. Comparable levels of gene silencing was observed between our shRNA-like siRNA (BBa_K3224002) and the shRNA (BBa_K3224001) construct we synthesized by referring the literature. (Wang et al., 2017)

Figure: Relative expression levels of surviving against GAPDH as control, effected by shRNA (adopted from literature) and shRNA-like siRNA (pur part) delivered by lipofectamine 2000 and CPP (our part).

Reference:

Wang YL, Shao X, Wang F, et al. Intron-specific shRNA-mediated downregulation of survivin and promotion of apoptosis in HeLa cells. Oncol Lett. 2017;14(5):5927–5933. doi:10.3892/ol.2017.6996