Figure 1.
Schematic presentation of SELEX-Seq strategy.
A, Oligonucleotides and PCR primers. The sequences underlined with arrows represented the annealing sites of primers. The bases at the 5′ end of nested primers represented the split barcodes. Biotin-labeled primers were used to prepare the biotin-labeled dsDNAs for NIRF-EMSA. B, EMSA-based SELEX-Seq procedure. The oligonucleotides in red, green and blue represented the positive, negative and random dsDNAs, respectively. PCR1, PCR2 and PCR3 were used to amplify the positive, negative and random dsDNAs with primers P-F/R, N-F/R and S-F1-4/R1-4, respectively. a, annealing; e, elongation; b, binding; p, purification; E, EMSA; P, PCR; R0 to R4, Round 0 to Round 4.
Figure 2.
Preparation and evaluation of dsDNAs used in SELEX-Seq.
A, Detection of dsDNA products of Klenow reaction with PAGE. B and C, Detection of purified dsDNA with PAGE before and after EB staining. The loading amount of dsDNA: 1, 4, 7: 1 µl; 2, 5, 8: 2 µl; 3, 6, 9: 4 µl. D and E, Detection of dsDNA with EMSA PAGE before and after EB staining. P, positive dsDNA; N, negative dsDNA; R, random dsDNA. Black arrowhead, shifted dsDNA (protein-bound dsDNA); Gray arrowhead, free dsDNA. All PAGE gels were visualized with UV transilluminator.
Figure 3.
Four rounds of EMSA-based SELEX selection and specificity detection.
A and B, Detection of EMSA PAGE gels before and after EB staining. The gel slices containing shifted random dsDNA were cut out before EB staining. Components of EMSA reactions were shown under the images. P, positive dsDNA; R, random dsDNA. Black arrowhead, shifted dsDNA; Gray arrowhead, free dsDNA. C, PCR detection of the SELEX-selected dsDNA. NC, PCR negative controls; N, negative dsDNAs. The selected random dsDNAs were amplified with the nested primers in various SELEX rounds (S-F1/R1 in R1, S-F2/R2 in R2, S-F3/R3 in R3, and S-F4/R4 in R4). R1 to R4, Round 1 to Round 4. D, Detection of the purified PCR products of random dsDNAs. M, DNA maker, the length of the smallest band is 100-bp. All PAGE gels were visualized with UV transilluminator.
Figure 4.
Evaluation of SELEX products with EMSA.
A, NIRF-EMSA detection of the selected random dsDNAs from each round. B, Signal quantification of shifted bands of Image A. Components of EMSA reactions were shown under the image. Black arrowhead, shifted DNA; Gray arrowhead, free DNA. R0 to R4, DNAs obtained from Round 0 to Round 4.
Figure 5.
Finding motifs with reads from Round 4 by using MEME.
Five motifs were extracted for each of five samples containing various numbers of non-overlapping 16-mer reads from Round 4. The top four motifs were displayed above the histogram of their fold enrichments. M1 to M4, Motif 1 to motif 4. FE, fold enrichment. The title of Y-axis of motif logos was “bits” and the labels were 0, 1 and 2. The labels under the motif logos were “1” to “10” from left to right that referred to the positions of nucleotides in a motif.
Figure 6.
Finding motifs with reads from various rounds by using MEME.
Five motifs were extracted for each of five samples containing 10000 non-overlapping reads from Round 1 to 4. The top four motifs were displayed above the histogram of their fold enrichments. M1 to M4, Motif 1 to motif 4. FE, fold enrichment. R1 to R4, Round 1 to Round 4. The title and labels of coordinate axes of motif logos were same as Figure 5.
Figure 7.
Intermediate and final motifs of NF-κB p50.
The intermediate motifs were the most enriched motifs obtained with 10 samples containing 10000 non-overlapping reads from Round 4. The final motifs were obtained with the reads containing 10 intermediate motifs. The known NF-κB motifs came from TRANSFAC, uPBM, custom PBM (cPBM). The title and labels of coordinate axes of motif logos were same as Figure 5.
Figure 8.
Evaluation of SELEX-Seq with known NF-κB motifs and EMSA-detected affinity.
A, Enrichment of NF-κB motifs were shown by the absolute number and the percent of reads that contained a particular motif in a round. The known NF-κB motifs came from TRANSFAC and JASPAR. B, Correlation of the fold enrichment of 10 sequences (10-mer) in four rounds with the relative affinity determined by EMSA. Because the EMSA value was less than 3.0, to compare with EMSA values, the logarithmic value of fold enrichment was calculated.