Biomod/2017/StJohns:Results

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Results

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.

All gel images have been uploaded in JPEG form, but TIFF forms can be made available upon request.


Effect of Trehalose on VLP Binding

Figure 23
Figure 23. Triangles behave as expected when bound with the new VLPs, as seen in lanes 6 and 7, unlike in lane 5 where old VLPs were used. There is a clear difference in movement of the VLP band when comparing the old with the new batch, in lanes 5 and 6. Using trehalose as a cryo-protectant does not seem to affect VLPs adversely, as there is no difference in behavior between fresh (lane 6) and frozen (lane 7).

1% Agarose gel; 650 Vhrs

Lanes

  1. 0.05 pmol Triangle blunt
  2. 0.05 pmol Triangle blunt + 0.20 pmol T21 VLP
  3. 0.05 pmol Triangle sticky
  4. 0.05 pmol Triangle sticky + 0.20 pmol wild type VLP
  5. 0.05 pmol Triangle sticky + 0.20 pmol old T21 VLP
  6. 0.05 pmol Triangle sticky + 0.20 pmol new T21 VLP (fresh)
  7. 0.05 pmol Triangle sticky + 0.20 pmol new T21 VLP (1 week frozen)


Tightening of Bands as a Result of Removing Hinge Staples

Figure 24
Figure 24. With the removal of ‘hinge staples,’ the DNA claws have much tighter bands on gels compared to previous versions.

1% Agarose; 700 Vhrs

Lanes:

  1. 0.05 pmol hinged claw blunt
  2. 0.05 pmol unhinged claw blunt
  3. 0.05 pmol hinged claw sticky
  4. 0.05 pmol unhinged claw sticky
Figure 24a
Figure 24a. A comparison of the old hinged claws (left) that gave T-shapes, and close-ups of the unhinged claws (center and right).

Distinction Between the Bound and Unbound Version of Unhinged Claw to VLP

Figure 25
Figure 25. With tighter bands for unhinged claws, a much more distinct change can be observed. The change from the unbound unhinged claw (lane 3) to the VLP-bound unhinged claw (lane 4) indicates that some kind of interaction has happened between the claws and the VLPs. No interaction is noticed for the blunt variety of unhinged claw (lanes 1 and 2), as expected.

1% Agarose; 690 Vhrs

Lanes:

  1. 0.05 pmol unhinged claw blunt
  2. 0.05 pmol unhinged claw blunt + 0.20 pmol T21 VLP
  3. 0.05 pmol unhinged claw sticky
  4. 0.05 pmol unhinged claw sticky + 0.20 pmol T21 VLP


FRET Signaling of Bound and Unbound Claw

Figure 26
Figure 26. The FRET channel scan of an illustrative gel indicating the problem we are having with FRET studies for our claws. As seen in lane 5, the unbound claw is displaying a FRET signal rather than the bound sample. The bound claw sample was expected to display a more intense FRET signal, as the donor and acceptor dyes are much closer together in the desired binding event. The donor used was ATTO-550, that excited at 554 nm and emitted at 576 nm. The acceptor used was ATTO-647N, that excited at 644 nm and emitted at 669 nm. In this binding event, the emitted donor light is used to excite the acceptor, which produces the FRET signal we expect to see.

1% Agarose; 680 Vhrs

Lanes:

  1. 0.05 pmol unhinged claw sticky (donor only)
  2. 0.05 pmol unhinged claw sticky (donor only) + 0.20 pmol T21 VLP
  3. 0.05 pmol unhinged claw sticky (acceptor only)
  4. 0.05 pmol unhinged claw sticky (acceptor only) + 0.20 pmol T21 VLP
  5. 0.05 pmol unhinged claw sticky (donor+acceptor)
  6. 0.05 pmol unhinged claw sticky (donor+acceptor) + 0.20 pmol T21 VLP

Yield Produced by Cross-Linking

Figure 27
Figure 27. Acrylamide gel reveals 30% of DNA strands denature in lane 9. This indicates that in this lane 70% of the triangle did not denature and was presumed to be still crosslinked. Lanes 2 and 6 show different concentrations of 8-MOP, and there was little to no difference in results. Lane 5 duplicates lane 9 with the sole difference of 8-MOP concentration.

20% Denaturing Acrylamide; 400 Vhrs

Lanes:

  1. Marker
  2. 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 0min)
  3. 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 30 min)
  4. 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 90 min)
  5. 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 270 min)
  6. 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 0 min)
  7. 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 30 min)
  8. 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 90 min)
  9. 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 270 min)
  10. Plasmid

Increased Reaction Time Led to Increased Crosslinking Yield

Figure 28
Figure 28. As seen by our calculated returns of crosslinked triangles, we concluded that increasing the reaction time of crosslinking increased the yield of our crosslinked triangles. We are unsure why higher concentrations of 8-MOP give lower yields.

Binding triangle with fluorescently labeled DNA strand 40%

Figure 29
Figure 29. Agarose gel showing crosslinked triangle binds to fluorescently labeled DNA strand. This indicates that our triangle was able to pick up the strands, indicating functionality. Lanes 1-3 indicate uncrosslinked triangle and lanes 4-6 indicates triangle becoming slightly heavier due to binding to DNA strand.

1% Agarose; 680 Vhrs

Lanes:

  1. ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
  2. ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
  3. ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
  4. ~0.05pm Crosslinked Triangle (270 min) + T21 Fluorescent Strand
  5. ~0.05pm Crosslinked Triangle (270 min) + T21 Fluorescent Strand
  6. ~0.05pm Crosslinked Triangle (480 min)+ T21 Fluorescent Strand


Purity Check and Quantifying Methylene Blue Strands

Figure 30
Figure 30. This is an unstained gel, allowing us to visualize MB's presence on DNA strands using the dye's intrinsic fluorescence (note that we haven't been able to find this anywhere in the literature). The purity check was successful revealing that lanes 2-7 have a single major band. The later dark band is caused by the dye used to detect how far the DNA has moved so it is irrelevant. A decrease in intensity is easily seen between the pairs of lanes (2,3), (4,5), and (6,7) which were loaded with 5 pmol, 0.5pmol, and 0.05 pmol respectively.


20% Acrylimide Gel; 794 Vhrs (Imaged using scan for MB before staining)


Lanes:

  1. 10 bp Ladder
  2. 5.0 pmol (TCA)7 MB
  3. 5.0 pmol T21 MB
  4. 0.5 pmol (TCA)7 MB
  5. 0.5 pmol T21 MB
  6. 0.05 pmol (TCA)7 MB
  7. 0.05 pmol T21 MB
  8. 5.0 pmol A21 C10
  9. 5.0 pmol (TCA)7 MS2
  10. 0.5 pmol Claw XII ATTO
  11. Denaturing Dye
  12. Blank

Quantifying Methylene Blue strands on Triangle

Prestain

Figure 31
Figure 31. (Imaged using scan for MB before staining) Binding with MB is seen in the shifted lanes of the stained image of the gel, and as expected no MB was observed in lanes that were incompatible (Ex. Lane 5- Triangle A21 + (TCA)7 MB). Additionally, a significant decrease in intensity is seen between the 15 sticky stranded triangle and 7 sticky stranded triangle.

1% Agarose Gel; 487 Vhrs (Imaged using scan for MB before staining)

Lanes:

  1. 1kb Ladder
  2. 0.05 pmol Triangle Blunt + 5.0 pmol T21 MB
  3. 0.05 pmol Triangle Blunt + 5.0 pmol (TCA)7 MB
  4. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
  5. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol (TCA)7 MB
  6. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol T21 MB
  7. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
  8. 0.05 pmol Up Triangle(7) A21 + 5.0 pmol T21 MB
  9. 0.05 pmol Up Triangle(7) A21 + 5.0 pmol (TCA)7 MB
  10. 0.05 pmol Up Triangle(7) (TGA)7 + 5.0 pmol T21 MB
  11. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB

SYBR Stain

Figure 32
Figure 32. (SYBR Stain) Successful Up Triangle synthesis with MB is observed. The gel shift between lanes 4 and 5 is evidence of a binding occurrence at the 15 sticky stranded triangle. A gel shift was also observed, but significantly less, with the 7 sticky stranded triangle. The lanes that observed shifting are labeled using red arrows (4,7,8,11).

1% Agarose Gel; 487 Vhrs (SYBR stain)


Lanes:

  1. 1kb Ladder
  2. 0.05 pmol Triangle Blunt + 5.0 pmol T21 MB
  3. 0.05 pmol Triangle Blunt + 5.0 pmol (TCA)7 MB
  4. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
  5. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol (TCA)7 MB
  6. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol T21 MB
  7. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
  8. 0.05 pmol Up Triangle(7) A21 + 5.0 pmol T21 MB
  9. 0.05 pmol Up Triangle(7) A21 + 5.0 pmol (TCA)7 MB
  10. 0.05 pmol Up Triangle(7) (TGA)7 + 5.0 pmol T21 MB
  11. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB

Attachment of MB strands to Side-Triangle

Prestain

Figure 33
Figure 33. (Imaged using scan for MB before staining) The shifted lanes 4 and 7 displayed MB attachment with similar intensities. As expected lanes 5 and 6 showed no sign of MB at the band region.

1% Agarose Gel; 581 Vhrs (Imaged using scan for MB before staining)


Lanes:

  1. 1kb Ladder
  2. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
  3. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
  4. 0.05 pmol Side Triangle(15) A21 + 5.0 pmol T21 MB
  5. 0.05 pmol Side Triangle(15) A21 + 5.0 pmol (TCA)7 MB
  6. 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol T21 MB
  7. 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB

SYBR Stain

Figure 34
Figure 34. (SYBR Stain) Side triangle with MB was synthesized forming a tight band in lanes 4 and 7. These lanes also displayed a shift in comparison to their corresponding non binding triangle in lanes 5 and 6 respectively.

1% Agarose Gel; 581 Vhrs (SYBR stain)


Lanes:

  1. 1kb Ladder
  2. 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
  3. 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
  4. 0.05 pmol Side Triangle(15) A21 + 5.0 pmol T21 MB
  5. 0.05 pmol Side Triangle(15) A21 + 5.0 pmol (TCA)7 MB
  6. 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol T21 MB
  7. 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB

Fluorescence Polarization Results

Figure 35
Figure 35. Repetition of standard dye Phloxine B with polarization value accurate according to literature value. Image reflects typical set of values; total of four dyes used to demonstrate polarization measurement ability.
Figure 36
Figure 36. Demonstrated repetition of polarization measurements using control variables. (Flouraptamer = short-hand fluorescent aptamer)
Figure 37
Figure 37. The blue curve represents fluorescent aptamer addition to NoV VLP, where the S-shaped curve indicates binding between the aptamer and the VLP. The orange curve represents T21 addition to NoV VLP, where the fairly constant polarization indicates no interaction between T21 and VLP. The NoV strand used for this binding experiment is GII.4.
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