Biomod/2013/Aarhus/Supplementary/Optimizations

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Optimizations

Optimization of click reactions of siRNA and sisiRNA

The protocols for the different click reactions were established using a 5’ hexynyle modified DNA oligo (5’-Hexynyl-GGTCATCCATGACAACTTTTT-3’). All optimization reactions were performed in a volume of 30 µL with 10 µL HEPES buffer titrated to the desired pH and 10 µL click buffer as listed in table 2. The remaining 10 µL of the total volume consisted of nucleotides and PEG or CPPs dissolved in dimethyl sulfoxide (DMSO), so that at least a third of the total volume was DMSO.

Table opt.1: Contents of the click buffer
Table opt.1: Contents of the click buffer

Optimization studies for PEGylations were performed with a PEG1K and the DNA oligo at three different pH values: 6.0, 7.4 and 8.0. The reactions were incubated at 50oC on the thermo shaker for 3 hours or 24 hours to establish the amount of time that was required for the reaction to take place. The concentrations of DNA used were 5 µM, 10 µM, 15 µM and 20 µM along with 2.5 or 5 equivalents of PEG. For the peptide GALA, (H-azidopentane acid-WAALAEALAEALAEHLAEALAEALEALAA, from GL Biochem Shanghai), the optimal reaction conditions were tested using 20 µM DNA oligo and 2.5 or 5 equivalents of peptide at three different pH values, 6.0, 7.4 and 8.0. The reactions were incubated for 6 hours at 50oC on the thermo shaker. Optimization reactions with the peptide melittin (H-azidopentane acid-IGAVLKVLTTGLPALISWIQQAQQL-OH, from Biosyntan) and the DNA oligo were made using 20 µM DNA oligo and 5 equivalents of peptide. Two reactions were carried out, at pH 7.4 and 8.0. Click reactions with the DNA oligo and PEG1K, that were made to establish the optimal reaction conditions, showed that the highest used concentration of oligo (20 µM) and 5 equivalents of PEG performed the best as seen in lanes 2 and 10, Figure 13. When comparing 3 hours of incubation time with 20 hours, no significant increase in reaction yield was observed, and 3 hours of incubation was therefore selected for the further reactions. The experiments that were conducted at pH 6.0 and 8.0 showed a slightly lower yield than at 7.4 (results not shown) and these conditions were therefore not used for the later PEGylations.

Figure opt.1 Optimization reaction at pH 7.4 with PEG1K and DNA oligo. 1) 25 bp DNA ladder, 2) 20 µM DNA, 5 x PEG1K, 3 hours incubation, 3) 20 µM DNA, 2.5 x PEG1K, 3 hours incubation, 4) 15 µM DNA, 5 x PEG1K, 3 hours incubation, 5) 15 µM DNA, 2.5 x PEG1K, 3 hours incubation, 6) 10 µM DNA, 5 x PEG1K, 3 hours incubation, 7) 10 µM DNA, 2.5 x PEG1K, 3 hours incubation, 8) 5 µM DNA, 5 x PEG1K, 3 hours incubation, 9) 5 µM DNA, 2.5 x PEG1K, 3 hours incubation, 10) 20 µM DNA, 5 x PEG1K, 20 hours incubation, 11) 20 µM DNA, 2.5 x PEG1K, 20 hours incubation, 12) 15 µM DNA, 5 x PEG1K, 20 hours incubation, 13) 15 µM DNA, 2.5 x PEG1K, 20 hours incubation, 14) 10 µM DNA, 5 x PEG1K, 20 hours incubation, 15) 10 µM DNA, 2.5 x PEG1K, 20 hours incubation, 16) 5 µM DNA, 5 x PEG1K, 20 hours incubation, 17) 5 M DNA, 2.5 x PEG1K, 20 hours incubation, 18) Control DNA oligo
Figure opt.1 Optimization reaction at pH 7.4 with PEG1K and DNA oligo. 1) 25 bp DNA ladder, 2) 20 µM DNA, 5 x PEG1K, 3 hours incubation, 3) 20 µM DNA, 2.5 x PEG1K, 3 hours incubation, 4) 15 µM DNA, 5 x PEG1K, 3 hours incubation, 5) 15 µM DNA, 2.5 x PEG1K, 3 hours incubation, 6) 10 µM DNA, 5 x PEG1K, 3 hours incubation, 7) 10 µM DNA, 2.5 x PEG1K, 3 hours incubation, 8) 5 µM DNA, 5 x PEG1K, 3 hours incubation, 9) 5 µM DNA, 2.5 x PEG1K, 3 hours incubation, 10) 20 µM DNA, 5 x PEG1K, 20 hours incubation, 11) 20 µM DNA, 2.5 x PEG1K, 20 hours incubation, 12) 15 µM DNA, 5 x PEG1K, 20 hours incubation, 13) 15 µM DNA, 2.5 x PEG1K, 20 hours incubation, 14) 10 µM DNA, 5 x PEG1K, 20 hours incubation, 15) 10 µM DNA, 2.5 x PEG1K, 20 hours incubation, 16) 5 µM DNA, 5 x PEG1K, 20 hours incubation, 17) 5 M DNA, 2.5 x PEG1K, 20 hours incubation, 18) Control DNA oligo

In the optimization reactions using GALA and DNA oligo, the reaction using 5 equivalents of peptide made at pH 8.0 appeared to give the highest yield as can be seen in lane 7 of Figure 14A. This correlates with the fact that the secondary structure of GALA is pH dependent, indicating that the peptide in random coil is more likely to react with the oligo than when secondary structure is present.

Figure opt2. A: Optimization reaction with GALA and DNA oligo. 1) 25 bp DNA ladder, 2) 20 µM DNA oligo, 2.5 x GALA, pH 6, 3) 20 µM DNA oligo, 5 x GALA, pH 6, 4) 20 µM DNA oligo, 2.5 x GALA, pH 7.4, 5) 20 µM DNA oligo, 5 x GALA, pH 7.4, 6) 20 µM DNA oligo, 2.5 x GALA, pH 8, 7) 20 µM DNA oligo, 5 x GALA, pH 8, 8) Control DNA oligo B: Optimization with DNA oligo and Melittin. 1) 25 bp DNA ladder,  2) 15-mer oligo, 3) 45-mer oligo, 4) 20 µM DNA oligo, 5 x melittin, pH 7.4, 5) 20 µM DNA oligo, 5 x melittin, pH 8, 6) Control DNA oligo.
Figure opt2. A: Optimization reaction with GALA and DNA oligo. 1) 25 bp DNA ladder, 2) 20 µM DNA oligo, 2.5 x GALA, pH 6, 3) 20 µM DNA oligo, 5 x GALA, pH 6, 4) 20 µM DNA oligo, 2.5 x GALA, pH 7.4, 5) 20 µM DNA oligo, 5 x GALA, pH 7.4, 6) 20 µM DNA oligo, 2.5 x GALA, pH 8, 7) 20 µM DNA oligo, 5 x GALA, pH 8, 8) Control DNA oligo B: Optimization with DNA oligo and Melittin. 1) 25 bp DNA ladder, 2) 15-mer oligo, 3) 45-mer oligo, 4) 20 µM DNA oligo, 5 x melittin, pH 7.4, 5) 20 µM DNA oligo, 5 x melittin, pH 8, 6) Control DNA oligo.

The optimization of the click reaction containing DNA oligo and melittin appeared to give the highest yield at pH 7.4 as seen in lane 4 of Figure 14B. This could also be due to the transition from random coil to α-helical structure that occurs at higher pH for this peptide. This suggests that the secondary structure inhibits the reaction to some degree, equivalent to the reaction with GALA.

Optimization of sisiRNA annealing reactions

Annealing of sisiRNA duplex The annealing of sisiRNA was tested using low concentrations of the strands in different ratios. The tests were run with a constant 0.25 µM concentration of antisense strand (W376) and variable concentrations of sense strands (W179 and W004). Following W376:W004:W179 ratios, given by the numbers in brackets, were tested: • Equal amounts of sense and antisense strands (1:1:1) • Excess of sense strands (1:1.5:1.5), (1:1.5:1.5 with TCEP) and (1:2:2) • Deficiency of sense strands (1:0.8) • Without one of the sense strands (1:1:0) and (1:0:1)

Previous annealings revealed it to be difficult to obtain complete annealing of the three strands; therefore we also tested for potential problems with reduction of the disulfide linker by reducing one of the old annealing reactions with 1000-fold molar excess TCEP (tris(2-carboxyethyl)phosphine) as a reducing agent. The duplexes were annealed using the preprogrammed annealing scheme in the PCR machine. 3.5 pmol of each sample with glycerol loading buffer added to a final concentration of 5% were run on a 12% native PAGE gel for 8.5 h at 120 V in 4° C.

Figure opt3. Annealing of different ratios of sisiRNA visualized on native PAGE. lane1: 25 bp DNA ladder; lane 2: annealing (1:1:1); lane 3: annealing (1:1.5:1.5); lane 4: annealing (1:1.5:1.5) + TCEP; lane 5: annealing (1:2:2); lane 6: annealing without W004 (1:1:0); lane 7: annealing without W179 (1:0:1); lane 8: control W004; lane 9: Control W179 C6 amino linker; lane : Control W179 + TCEP; lane 11: Control W376
Figure opt3. Annealing of different ratios of sisiRNA visualized on native PAGE. lane1: 25 bp DNA ladder; lane 2: annealing (1:1:1); lane 3: annealing (1:1.5:1.5); lane 4: annealing (1:1.5:1.5) + TCEP; lane 5: annealing (1:2:2); lane 6: annealing without W004 (1:1:0); lane 7: annealing without W179 (1:0:1); lane 8: control W004; lane 9: Control W179 C6 amino linker; lane : Control W179 + TCEP; lane 11: Control W376

No improvement was shown compared with earlier annealing reactions (not shown) when treating with TCEP (Figure 23, lane 4), suggesting that the disulfide linkers cannot be blamed for the annealing problems. The experiment suggests that a duplex cannot be formed if one of the sense strands is missing, apparently because of stacking interactions. Thus the amount of sense strands should be in equal amount. It appears that the reactions done with a 1:1:1 and a 1:1.5:1.5 ratio of W376:W004:W179 worked better (Figure 23, lane 2 and 3). Future annealing reactions were done with a ratio of 1:1.3:1.3.


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