Biomod/2015/StJohns:Results

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Results

Figure 11: Optimization methods for NClaw

Test # DNA on Claw DNA on MS2 Capsid Capsid: Claw ratio Mg2+ conc. (mM) Temperature (°C)
1 A21 T21 2:1 ~10 Room temp (RT)
2 A21 T21 8:1 ~10 RT
3 A21 T21 44:1 ~24 RT
4 A21 T21 10:1 ~24 RT
5 A21 T21 1:1 ~24 RT
6 A21 T21 1:10 ~24 RT
7 A21 T21 10:1 ~24 4
8 A21 T21 1:1 ~24 4
9 A21 T21 1:10 ~24 4
10 A21 T21 10:1 ~24 15
11 A21 T21 1:1 ~24 15
12 A21 T21 1:10 ~24 15
13 A21 T21 44:1 ~24 15
14 A21 T21 10:1 ~48 RT
15 A21 T21 40:1 ~24 RT
16 A21 T21 30:1 ~24 RT
17 A21 T21 20:1 ~24 RT
18 A21 T21 40:1 ~20 RT
19 A21 T21 40:1 ~10 RT
20 A21 T21 40:1 ~5 RT
21 A21 T21 40:1 ~2.5 RT
22 A21 T21 40:1 ~20 (with 1M urea) RT
23 A21 T21 40:1 ~10 (with 1M urea) RT
24 (TGA)7 (TCA)7 10:1 ~20 RT
25 (TGA)7 (TCA)7 10:1 ~10 RT
26 (TGA)7 (TCA)7 10:1 ~5 RT
27 (TGA)7 (TCA)7 10:1 ~2.5 RT
28 (TGA)7 (TCA)7 20:1 ~20 RT
29 (TGA)7 (TCA)7 20:1 ~2.5 RT
30 (TGA)7 (TCA)7 30:1 ~2.5 RT
31 (TGA)7 (TCA)7 50:1 ~2.5 RT

NOTE:

Unless otherwise stated, the agarose gels run (Fig. 12-19) were 0.5X TBE ~10mM Mg2+ 1% denaturing, with 0.5X TBE 10mM Mg2+ buffer, run at 25°C and 450-700 volthours.

June 9 2015: Diff ratio MS2T21 gel

Figure 12-NClaw and Capsid, Varying stoichiometric ratios Gel
Figure 12-NClaw and Capsid, Varying stoichiometric ratios Gel. Image representative of all prior gels with binding experiments (Fig 11). No band shift occurs for sticky NClaw with MS2T21, but there is significant band smearing for those lanes.

Lanes:
(Claw: 1 equivalent = 0.1 pmol)

  1. 1kb DNA marker
  2. Vanilla Claw Blunt (VB)
  3. Vanilla Claw Sticky (VS)
  4. T21 modified MS2 virus capsids (MS2T21)
  5. MS2T21:VB, 1:1
  6. MS2T21:VS, 1:1
  7. MS2T21:VB, 2:1
  8. MS2T21:VS, 2:1
  9. MS2T21:VB, 5:1
  10. MS2T21:VS, 5:1
  11. MS2T21:VB, 10:1
  12. MS2T21:VS, 10:1

June 30 2015: Triangle binding gel

Figure 13-DO Triangle Gel. Successful band shift for Triangle Sticky with MS2T21
Figure 13-DO Triangle Gel. Successful band shift for Triangle Sticky with MS2T21

Lanes:
(Triangle: 1 equivalent = 0.05 pmol)

  1. 1kb DNA marker
  2. Triangle Blunt
  3. Triangle Sticky
  4. MS2T21
  5. MS2T21:Triangle Blunt, 4:1
  6. MS2T21:Triangle Sticky, 4:1

July 9 2015: NClaw 5S and 4S binding

Figure 14-Multiple binding sites per arm Gel
Figure 14-Multiple binding sites per arm Gel. When annealed with MS2T21, less NClaw 5S and NClaw 4S remains as free claws (unbound claws). Those lanes also have material left in the wells that did not travel through the gel, signifying polymerization.

Lanes:
(Claw: 1 equivalent = 0.05 pmol)

  1. 1kb DNA marker
  2. NClaw Blunt
  3. MS2T21:NClaw Blunt, 4:1
  4. NClaw 5 Sticky/arm (NClaw 5S)
  5. Blunt MS2:NClaw 5S, 4:1
  6. MS2T21:NClaw 5S, 4:1
  7. NClaw 4 Sticky/arm (NClaw 4S)
  8. Blunt MS2:NClaw 4S, 4:1
  9. MS2T21:NClaw 4S, 4:1

July 16 2015: TClaw Synthesis and polymerization Gel

Figure 15-TClaw Synthesis and polymerization Gel
Figure 15-TClaw Synthesis and polymerization Gel. Figure 15 shows a gel image demonstrating a gel shift Plasmids to TClaw, indicating a conformational change. Nonetheless, there is polymerization indicating that the Tethers are causing the claws to aggregate together. This is not favorable for capsid binding.

Date: 07.16.15;
Voltage: 70 Volts/Hour; Length of Run: 4 H 31M Concentration of Sample: 19.23nanoM: Blunt T22 Claw and Sticky T22 Claw; 20.00nanoM Blunt T31 Claw; 20.83nanoM: Sticky T31 Claw; 18.87nanoM: Blunt T40 Claw and Sticky T40 Claw; 18.52nanoM: Blunt T49 Claw; 17.86nanoM: Sticky T49 Claw Name of each sample:

  1. Blunt T40 Claw
  2. Sticky T40 Claw
  3. Blunt T49 Claw
  4. Sticky T49 Claw
  5. Blunt T22 Claw
  6. Sticky T22 Claw
  7. Blunt T31 Claw
  8. Sticky T31 Claw
  9. Blank
  10. Plasmid
  11. 1KB Ladder

Quantity of DNA in each sample: 0.05 picomoles

July 30 2015: TClaw Synthesis and polymerization Gel

Figure 16-TClaw synthesis less polymerization Gel
Figure 16-TClaw synthesis less polymerization Gel. Figure 16 shows gel where TClaws were made using different annealing protocol (See Approach). TClaws show much less polymerization here, compared to Figure 15, indicating that tethers might be binding to desired locations.

Date: 06.30.15;
Voltage: 70 Volts/Hour; Length of Run: 5 H 20M Concentration of Sample: 10.20nanoM: Blunt T Claw; 10.64nanoM Sticky T Claw; 0.3uM Tether Name of each sample:

  1. Blunt Claw
  2. Sticky Claw
  3. Blunt Claw, 22nm Tether
  4. Sticky Claw, 22nm Tether
  5. Blunt Claw, 31 nm Tether
  6. Sticky Claw, 31 nm Tether
  7. Blunt Claw, 40nm Tether
  8. Sticky Claw, 40nm Tether
  9. Blunt Claw, 49nm Tether
  10. Sticky Claw 49nm Tether
  11. Blank
  12. 1 KB Marker

Quantity of DNA in each sample: 0.05 picomoles Claw; 0.25 picomoles of Tether; 9 picomoles Hex

August 12 2015: Post SYBR stain

Figure 17-TClaw and Hex Strands SYBR stain Gel
Figure 17-TClaw and Hex Strands SYBR stain Gel. Figure 17 Shows an image of the same TClaw made in figure 16, this time with an of excess HEX strand (Complementary to tether) and excess tether.

Date: 08.12.15; Voltage: 70 Volts/Hour; Length of Run: 4 H 12M Concentration of Sample: 18.18nanoM: Blunt T Claw; 19.05nanoM Sticky T Claw; 0.2uM Tether; 4.5uMHex Name of each sample:

  • Blunt T31 Claw+ Hex
  • Sticky T31 Claw + Hex
  • Blunt T40 Claw + Hex
  • Sticky T40 Claw + Hex
  • Blunt T 49 Claw + Hex
  • Sticky T49 Claw +Hex
  • Blunt NClaw (No tether) + Hex
  • Sticky NClaw (No tether) + Hex
  • 1KB

Quantity of DNA in each sample: 0.05 picomoles Claw; 0.25 picomoles of Tether; 9 picomoles Hex

Figure 18: Pre-SYBR staining using Fluorescent Hex Strand

Figure 18-TClaw and Hex Pre-Sybr Gel
Figure 18-TClaw and Hex Pre-Sybr Gel. Figure 18 shows same gel as Figure 17 before staining with SYBR. HEX strands and tethers are visible. Lanes 1-4 (TClaw) have less tethers present in the product, when compared to lanes 5-6 (NClaw), potentially because tethers are binding to the claw, and just excess tether can be seen in lanes 1-4.

Date: 08.31.15; Voltage: 70 Volts/Hour; Length of Run: 4 H 0M. Concentration of Sample: 18.18nanoM: Blunt T Claw; 19.05nanoM Sticky T Claw; 0.2uM Tether; 0.2uM CY3 Name of each sample:

  1. Blunt T31 Claw+ Hex
  2. Sticky T31 Claw + Hex
  3. Blunt T40 Claw + Hex
  4. Sticky T40 Claw + Hex
  5. Blunt T 49 Claw + Hex
  6. Sticky T49 Claw +Hex
  7. Blunt NClaw (No tether) + Hex
  8. Sticky NClaw (No tether) + Hex
  9. 1KB

Quantity of DNA in each sample: 0.120 picomoles Claw; 3 picomoles of Tether; 10.8 picomoles Cy3

Figure 18: Post SYBR stain

Figure 18-TClaw and CY3 Sybr Stain Gel
Figure 18-TClaw and CY3 Sybr Stain Gel. Figure 18 shows TClaw with excess tethers and excess CY3 strand.

Figure 19: Pre-SYBR image using CY3 Fluorescent Strand

Figure 19-TClaw and CY3 pre-Sybr Gel
Figure 19-TClaw and CY3 pre-Sybr Gel. Figure 19 shows the same gel image as Figure 18 before staining with Sybr. CY3 strand has a lower LOD than the HEX strand used in the previous trials. In this figure we can clearly see that CY3 is binding to tether, and tether is binding to claw. Little polymerization indicates that there is less aggregation between the claws.

Figure 20: AFM image of NClaw

  • Figure 20: AFM image of NClaw. AFM images of NClaw showing good formation.

    Figure 20: AFM image of NClaw. AFM images of NClaw showing good formation.

  • Zoomed in NClaw

    Zoomed in NClaw

Fab’ Results and Discussion

Unless otherwise stated for Figures 21-26, the Gel Type: SDS PAGE; Percentage: 12% Gel; Buffer Type: 1X SDS Buffer; Temperature: 25C. Stained in Oriole Stain and ran at 300V.

  • Figure 21: Shows 3 different conditions for reducing Fab2’ to Fab’. Lane 3 (50mM 37°C 1hr) had the fewest amount of by-products and was continually used from then on instead of lane 4(50mM RT 1hr) and 5(1.6mM, 37°C, 1hr)

    Figure 21: Shows 3 different conditions for reducing Fab2’ to Fab’. Lane 3 (50mM 37°C 1hr) had the fewest amount of by-products and was continually used from then on instead of lane 4(50mM RT 1hr) and 5(1.6mM, 37°C, 1hr)

  • Figure 22-Shows smearing upward of where Fab’ is usually shown, indicative of reactivity. PageRuler, IgG2a, Fab’, Fab’ and PEG maleimide rxn.

    Figure 22-Shows smearing upward of where Fab’ is usually shown, indicative of reactivity. PageRuler, IgG2a, Fab’, Fab’ and PEG maleimide rxn.

  • Figure 23-Shows the smearing further up on the gel from where the DNA-Maleimide alone is shown, which proves the reactivity of the DNA. This gel is 20% Denaturing Acrylamide gel with 1X TBE Buffer at 65°C ran at 500V for 100min. Stained in SYBR for 15min.

    Figure 23-Shows the smearing further up on the gel from where the DNA-Maleimide alone is shown, which proves the reactivity of the DNA. This gel is 20% Denaturing Acrylamide gel with 1X TBE Buffer at 65°C ran at 500V for 100min. Stained in SYBR for 15min.

  • Figure 24-Shows the digestion and reduction of IgG1 to Fab’ and surrounding the Fab’ band on the gel, there are visibly more by-products.

    Figure 24-Shows the digestion and reduction of IgG1 to Fab’ and surrounding the Fab’ band on the gel, there are visibly more by-products.

  • Figure 26-Shows the first visible conjugation on a gel.

    Figure 26-Shows the first visible conjugation on a gel.

Figure 25:Trials with Pepsin and IgG2a shown, not including other enzyme attempts or antibodies

Test # Duration of conjugation Humidity Concentration Temperature Ratio of DNA to Fab’ AAA or TCA Conjugation?
1 3hr - 8ug/ul RT 5:1 both No
2 19hr - 1ug/ul RT 4:1 both No
3 3hr less 10ug/ul RT 150:1 both No
4 3hr - 10ug/ul RT 188:1 both No
5 3hr 30min less 10ug/ul RT 250:1 AAA Yes
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