Optimizations of SiRNA Design for the Activation of Gene Transcription by Targeting the TATA-Box Motif

<div><p>Small interfering RNAs (siRNAs) are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs) could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a) complementary to the TATA-box-centered region; (b) UA usage at the first two bases of the antisense strand; (c) twenty-three nucleotides (nts) in length; (d) 2′-O-Methyl (2′-OMe) modification at the 3′ terminus of the antisense strand; (e) avoiding mismatches at the 3′ end of the antisense strand. The optimized activating siRNAs potently enhance the expression of <i>interleukin-2</i> (<i>IL-2</i>) gene in human and mouse primary CD4⁺ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.</p></div

Sequence characters and length are correlated with the efficiency of siRNA-induced activation.

<p>(<b>A</b>) The frequency distributions of functional activating siRNAs with 1-A/U, 2-A/U or both in the antisense strand. ND, not detected. (<b>B</b>) Effects of sequence modification in the 5′ end of antisense strand on the siRNA-induced activation of <i>IL-2</i> 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. (<b>C</b>) Effects of siRNAs with different lengths on <i>IL-2</i> 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. <i>P</i>-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, <i>p</i><0.05. **, <i>p</i><0.01. ***, <i>p</i><0.001.</p

Optimized siRNAs showed higher and longer efficiency on <i>IL-2</i> promoter activation in human or mouse primary CD4⁺ T cells.

<p><b>(A)</b> HEK293T cells were transfected with siRNAs at the indicated concentrations and promoters activities were determined as described above. <b>(B)</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. <b>(C)</b> Western blot analysis of IL-2 protein in human primary CD4⁺ T cells in (B). The β-actin was selected as an internal control. <b>(D)</b> 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. <i>P</i>-values were calculated using the two tailed unpaired Student’s t-test with equal variances. *, <i>p</i><0.05. **, <i>p</i><0.01. ***, <i>p</i><0.001. These data represented three independent experiments.</p

Sequence mutations in the siRNA IL2-CEN or INS-CEN.

<p>Single (s) and double (ds) mutants were all named according to the position (from the 5′ end of the antisense strand) of the mutation. All mutations (in bold lowercase) were GC inversions relative to wild-type (wt), except that ds11/12 of IL2-CEN and ds10/11 of INS-CEN were mutated in the TATA-box.</p><p>Sequence mutations in the siRNA IL2-CEN or INS-CEN.</p

Mismatch tolerance of functional activating siRNAs.

<p>Promoter activities of (<b>A</b>) <i>IL-2</i> or (<b>B</b>) <i>INS</i> were determined with siRNAs harboring a serial mutations listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108253#pone-0108253-t002" target="_blank">Table 2</a>. The promoter activities were determined with dual-luciferase assay as described above. <i>P</i>-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, <i>p</i><0.05. **, <i>p</i><0.01. ***, <i>p</i><0.001.</p

Sequences of synthesized oligonucleotides targeting human <i>IL-2</i>, <i>insulin</i> (<i>INS</i>) or mouse <i>IL-2</i> promoter.

<p>For each siRNA, just the sense strand is shown. Nucleotides in bold were corresponding to TATA-box motif. Modified bases were in bold lowercase. ms, mouse.</p><p>Sequences of synthesized oligonucleotides targeting human <i>IL-2</i>, <i>insulin</i> (<i>INS</i>) or mouse <i>IL-2</i> promoter.</p

SiRNAs targeting TATA-box enhance the promoter activities specifically and efficiently.

<p>(<b>A</b>) Effects of siRNAs targeting TATA-box motif on promoter activities of 16 genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108253#pone-0108253-g001" target="_blank">Figure 1</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108253#pone.0108253.s001" target="_blank">File S1</a>. 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>B</b>) Mutations were introduced into the target site in <i>IL-2</i> core promoter and generated the mutated <i>IL-2</i> promoter, IL-2mt-27/−25 or IL-2mt-17/−16. HEK293T cells were transfected with the wild-type or mutated <i>IL-2</i> promoter-driven reporter constructs and siRNA IL2-CEN or NC. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. (<b>C</b>–<b>F</b>) SiRNAs targeting the promoter of <i>IL-2</i>, <i>INS</i>, <i>CIRBP</i>, <i>APOE</i>, <i>BCL2L12</i>, <i>c-Myc</i>, <i>POMC</i> or <i>GAPD</i> were co-transfected into HEK293T cells with (<b>C</b>) <i>IL-2</i>, (<b>D</b>) <i>BCL2L12</i>, (<b>E</b>) <i>POMC</i> or (<b>F</b>) <i>c-Myc</i> promoter-driven reporter constructs. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. n = 3. **, <i>p</i><0.01. ***, <i>p</i><0.001.</p

Certain chemical modifications enhance the siRNAs efficacy on gene promoter activation.

<p><b>(A</b>–<b>B</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 (<b>A</b>) 2 days or (<b>B</b>) 4 days post transfection. IL-2 mRNA levels were then evaluated by qRT-PCR and normalized to β-actin. <i>P</i>-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, <i>p</i><0.05.</p

TATA-box motif targeted siRNAs activate the promoter activities of 16 genes.

<p>(<b>A</b>) 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>B</b>–<b>Q</b>) 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. <i>P</i>-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, <i>p</i><0.05. **, <i>p</i><0.01. ***, <i>p</i><0.001.</p

Correlation between E-cadherin, vimentin and lymph node metastasis and p120ctn.

<p>Correlation between E-cadherin, vimentin and lymph node metastasis and p120ctn.</p