Table 1.
Sequences of synthesized oligonucleotides targeting human IL-2, insulin (INS) or mouse IL-2 promoter.
Table 2.
Sequence mutations in the siRNA IL2-CEN or INS-CEN.
Table 3.
Chemical modifications of siRNAs targeting the IL-2 TATA-box.
Figure 1.
TATA-box motif targeted siRNAs activate the promoter activities of 16 genes.
(A) Schematic diagram for designing siRNAs that target the TATA-box motif. The consensus sequence of TATA-box motif was highlighted. SiRNAs targeting the TATA-box-centered position (CEN), siRNAs targeting the positions in upstream (UP) or downstream (DN) of the TATA-box were indicated. (B–Q) Analyses of promoter activities of 16 genes with siRNAs targeting different sites of the TATA-box motif region. The indicated siRNAs were co-transfected with the target promoter-driven firefly luciferase (FL) and renilla luciferase (RL) constructs into HEK293T cells. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. NC, negative control siRNA. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001.
Figure 2.
SiRNAs targeting TATA-box enhance the promoter activities specifically and efficiently.
(A) Effects of siRNAs targeting TATA-box motif on promoter activities of 16 genes in Figure 1. The most effective siRNA for each gene was listed in the y-axis, and the promoter activities relative to NC were listed in the x-axis. These genes and functional activating siRNAs were classified according to the enhancement of siRNAs on promoter activities compared with NC (E (%)) as shown in Table S1 in File S1. Promoter activities were determined with dual-luciferase assay as described above. The frequencies of genes in three groups were: E>200, 37.5%; 200>E>100, 12.5%; 100>E>30, 50%. The frequencies of siRNAs CEN in each group were: E>200, 83.3%; 200>E>100, 100%; 100>E>30, 37.5%. (B) Mutations were introduced into the target site in IL-2 core promoter and generated the mutated IL-2 promoter, IL-2mt-27/−25 or IL-2mt-17/−16. HEK293T cells were transfected with the wild-type or mutated IL-2 promoter-driven reporter constructs and siRNA IL2-CEN or NC. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. (C–F) SiRNAs targeting the promoter of IL-2, INS, CIRBP, APOE, BCL2L12, c-Myc, POMC or GAPD were co-transfected into HEK293T cells with (C) IL-2, (D) BCL2L12, (E) POMC or (F) c-Myc promoter-driven reporter constructs. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. n = 3. **, p<0.01. ***, p<0.001.
Figure 3.
SiRNAs targeting the center of TATA-box motif upregulate gene promoter activities with high efficiency.
(A) The frequency distributions of functional activating siRNAs targeting different positions relative to the TATA-box. The consensus sequence of TATA-box motif was highlighted in the schematic diagram, and relative positions of siRNAs were indicated by the distances between the 3′ ends of siRNAs antisense strands and the 5′ ends of TATA-box (position 0). For example, the relative position of siRNA whose 3′ end targeting the third nucleotide of the TATA-box was position 2, and the relative position of siRNA CEN whose 3′ end targeting the sixth or fifth nucleotide upstream the TATA-box was position −6 or −5 (top panel). According to the promoter activities determined by dual-luciferase assay, functional activating siRNAs were divided into three groups (E>200, black square; 200>E>100, gray square; and 100>E>30, white square. Here E (%) stands for the enhancement of siRNA on promoter activity in percent compared with NC). The frequency distributions of the siRNAs with different relative positions in each group were indicated. ND, not detected. (B–D) Promoter activity assay of (B) IL-2, (C) INS or (D) APOE promoter with siRNAs against different sites of the TATA-box motif. SiRNAs targeting various positions centered with the TATA-box motif were transfected and promoters activities were determined as described above. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001.
Figure 4.
Sequence characters and length are correlated with the efficiency of siRNA-induced activation.
(A) The frequency distributions of functional activating siRNAs with 1-A/U, 2-A/U or both in the antisense strand. ND, not detected. (B) Effects of sequence modification in the 5′ end of antisense strand on the siRNA-induced activation of IL-2 promoter. 5u, the sequence-modified siRNA whose first base in 5′ end of antisense strand was substituted into U; 5ua, the sequence-modified siRNA whose first two bases in 5′ end of antisense strand were substituted into UA. (C) Effects of siRNAs with different lengths on IL-2 promoter activity. 5ua-15, the length-modified siRNA 5ua whose length was 15 nts; 5ua-19, the length-modified siRNA 5ua whose length was traditional 19 nts; and so on. The promoter activities were determined with dual-luciferase assay as described above. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001.
Figure 5.
Mismatch tolerance of functional activating siRNAs.
Promoter activities of (A) IL-2 or (B) INS were determined with siRNAs harboring a serial mutations listed in Table 2. The promoter activities were determined with dual-luciferase assay as described above. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001.
Figure 6.
Certain chemical modifications enhance the siRNAs efficacy on gene promoter activation.
(A–B) Effects of chemical modified siRNAs on IL-2 mRNA level in Jurkat cell line. 2–3×104 Jurkat cells were transfected with 120 pmol siRNAs for 12 hrs, and were subsequently treated with PMA (50 ng/ml) and ionomycin (1 µM) till (A) 2 days or (B) 4 days post transfection. IL-2 mRNA levels were then evaluated by qRT-PCR and normalized to β-actin. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05.
Figure 7.
Optimized siRNAs showed higher and longer efficiency on IL-2 promoter activation in human or mouse primary CD4+ T cells.
(A) HEK293T cells were transfected with siRNAs at the indicated concentrations and promoters activities were determined as described above. (B) Effects of optimized siRNAs on IL-2 mRNA level in human primary CD4+ T cells. Human primary CD4+ T cells were transfected with 120 pmol siRNAs for 12 hrs, and were subsequently stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (5 µg/ml) antibodies for 84 hrs. IL-2 mRNA levels were determined by qRT-PCR as described above. (C) Western blot analysis of IL-2 protein in human primary CD4+ T cells in (B). The β-actin was selected as an internal control. (D) Effects of optimized siRNAs on IL-2 mRNA level in mouse primary CD4+ T cells. Mouse primary CD4+ T cells were transfected with 120 pmol siRNAs for 12 hrs, and were subsequently stimulated with anti-mouse CD3 (2 µg/ml) and anti-mouse CD28 (1 µg/ml) antibodies for 84 hrs. Mouse IL-2 mRNA levels were determined by qRT-PCR and normalized to mouse GAPDH. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. *, p<0.05. **, p<0.01. ***, p<0.001. These data represented three independent experiments.
Table 4.
Contributions of five characteristics to the functionality of activating siRNAs.