IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-19

Protection assay

Incubation and dilution protocol, take 2

• Goal: to see the lower threshold of imaging streptavidin bound to biotin
• and to see if we can image any streptavidin-biotin construct on a gel at all
• Protocol: mix 6 μL 2 μM streptavidin with 6 μL 1 μM c.3.2.7.2b oligo, to give a final oligo concentration of 500 nM, and incubate at room temperature for 5 min.
• dilution and electrophoresis:
• each lane contained 2 μL of different dilutions (as per table below), 6 μL of water, and 2 μL of loading dye
  • lanes 7-12 were loaded with the same mixtures as lanes 1-6, respectively

lane	amt of 1x strep-biotin mix (μL)	dilution	H2O added (μL)	final oligo concentration (nM)	amt of oligo in 2 μL diluted mix (fmols)
3	2	1x	0	500	1000
4	1	5x	4	100	250
5	1	10x	9	50	100
6	1	40x	39	12.5	25
1	control: 1 μL 2 μM streptavidin (control) to give 2000 fmols streptavidin
2	control: 2 μL 1 μM oligo (control) to give 2000 fmols oligo

12% native PAGE. Lower detection threshold of streptavidin is 500 fmols (lane 3).

• ran on native polyacrylamide gel for 30 min. at 120V
• no DNA imaged with EtBr staining
• GelCode Blue staining shows a band in lane 3 with the same motility as control streptavidin in lane 1
  • this is probably free streptavidin
• unclear where streptavidin-biotin complex is
• expt appears to be consistent with manufacturer's notes that the lower detection limit is 8 ng (151 fmols streptavidin, MW=52.8 kDa) [1]

Imaging biotinylated oligos

• goal: show that we can image biotinylated oligos on a PA gel

lane	non-biotinylated DNA (Lewis' S5) (2 μM) (μL)	biotyinylated DNA (3.2.7.2b) (1 μM)	H2O (μL)	Tris-glycine loading dye (10x) (μL)	total amt of DNA (fmols)
1	3	0	5	2	6000
2	1	0	7	2	2000
3	0	5	3	2	5000
4	0	3	5	2	3000
5	0	1	7	2	1000
6	0	0.5	7.5	2	5000

12% native PAGE. Lower detection limit for biotinylated oligos is 3000 fmols (lane 4).

• run on a 12% native PA gel at 120V for 15 min.
• clear bands appear in lanes 1-4
• faint bands in lanes 5-6 are likely artifacts, as lanes 7-12 (empty) contain them as well

Brainstorming for future assay

Run two experiments in parallel, the first a control.

1. assmebly
   • assemble a nanostructure with outward- (1) and inward- (2) facing biotinylated oligos
   • incubate assembled nanostructures with fluorescently-labeled streptavidin
   • close the container lids
2. baseline protein assay
   • precipitate nanostructures
   • pour off supernatant (containing excess streptavidin)
   • resuspend nanostructures in water
   • measure streptavidin concentrations in each container (pre-digest)
     • these are baseline values, and we expect that they should be similar if incubation efficencies for each nanostructure are similar
3. protease digest
   • digest each nanostructure with protease
     • particular protease and protocol must be finalized
4. measure streptavidin concentrations in each container (post-digest)
   • these values should differ if biotinylated streptavidin can still be digested (container 1) and if a closed nanostructure protects its cargo (container 2)
5. confirmation of presence of streptavidin
   • treat both containers with DNAse
   • measure streptavidin concentrations
     • streptavidin is expected to be bound to biotin, but biotin not bound to oligos
   • treat both containers with protease
   • measure streptavidin concentrations
     • values should all be close to zero

Container 4.0

Reagents (each expt in respective 0.2 mL PCR tube)

Experiment	Scaffold	Oligos	Folding buffer	dH2O	total volume
-latches -aptamers	9 μL p7308	16 μL xxx (250 nm)	4 μL 10x	11 μL	40 μL
+latch1 -aptamers	9 μL p7308	16 μL xxx (250 nm)	4 μL 10x	11 μL	40 μL
+latch2 -aptamers	9 μL p7308	16 μL xxx (250 nm)	4 μL 10x	11 μL	40 μL
6 hb	9 μL p7308	4 μL 6hb.v5 (0.99 μM)	4 μL 10x	23 μL	40 μL
-oligos	9 μL p7308	-	4 μL 10x	27 μL	40 μL
-scaffold	-	16 μL xxx (250 nm)	4 μL 10x	20 μL	40 μL

Annealing protocol

• start at 80[[:Category:{{{1}}}|{{{1}}}]]
• 60 cycles: wait 2 minutes, decrease 1[[:Category:{{{1}}}|{{{1}}}]]
• hold at 4[[:Category:{{{1}}}|{{{1}}}]]

Gel analysis

• 2% agarose gel supplemented to 10 mM MgCl₂ and with 3 μL 10 mg/mL EtBr (100 mL gel)
• run in 1x TBE supplemented to 10 mM MgCl₂
• 45 min at 130 V

Lane	Contents	Loading Buffer (10x TBE/glycerol)
1	1kb DNA ladder (10 μL)	1.1 μL
2	(-)	1.1 μL
3	Ib (10 μL)	1.1 μL
4	4.0 -latches -aptamers (10 μL)	1.1 μL
5	4.0 +latch1 -aptamers (10 μL)	1.1 μL
6	4.0 +latch2 -aptamers (10 μL)	1.1 μL
7	6hb (10 μL)	1.1 μL
8	4.0 -oligos (10 μL)	1.1 μL
9	4.0 -scaffold	1.1 μL

• results

2% agarose gel electrophoresis. All folded structures run slower than scaffold alone.