IV. ParMRC and Expandable DNA Nanostructures
IV.1. Introduction
IV.1.1. Using DNA origami to study biophysical questions
IV.1.1.3. DNA origami design: proposed and actual
Our initial goal in this investigation of biophysics of the ParMRC system was to try to answer some questions in regards to the structure, orientation, and binding mode of a ParM filament with a ParR/parC complex, described in the previous section, by using DNA origami as a custom single-molecule template. We can create a DNA origami structure that has a parC sequence incorporated into its structure (Figure IV-4ab). By changing the subset of staple strands, we can easily vary parameters such as the position of the parC strand, the angle of the parC strand relative to the origami frame, and the distance between the ends of the parC sequence, therefore changing the tension of the parC strand. Then, we hoped we could study the interaction of the geometrically- constrained parC component with ParR and ParM. For example, if a ParR/parC complex can stay rigid enough on an origami template, we could measure the angle distribution of a ParM filament relative to the ParR/parC complex on the origami by taking AFM images, which may allow us to tell if ParM filaments associate with ParR/parC complexes at a preferred relative angle, perhaps either perpendicular or parallel (Figure IV-4c). Also, if we combine two origami structures together each containing a ParR/parC complex, controlling the bonds between the origami structures in such a way that we have multiple combinations of the orientation of the ParR/parC complexes (Figure IV-4d), we may be able to see different growth directions of a ParM filament, if there indeed exists polarity in the ParR/parC complexes. Further, depending on the orientation and alignment of the
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i
x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment
hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry of parC-ParR binding to ParM filaments.
10
Parallel orientation
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i
x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment
hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry of parC-ParR binding to ParM filaments.
10
Perpendicular orientation
“Wrap-around” model “Open-clamp” model
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i
x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment
hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC -ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry of parC-ParR binding to ParM filaments.
10
Polarized
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i
x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment
hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC -ParR orientation effects.
ParR
Figure 4: Characterizing the geometry of parC-ParR binding to ParM filaments.
10
Symmetric
(a)
(b)
(c)
84
ParR/parC complexes relative to the ParM filament growth direction, and on the force generated by the filament growth, we may be able to see an interesting behavior that the growing filament may push the two combined origami rectangles apart, which leads us to the idea of building ‘expandable’nanostructures described in the next section.
For our investigation, to achieve a DNA origami structure that contains a single parC sequence in it, we first created an M13-variant scaffold strand by genetically engineering M13 (via Genestitute) to include the 169-nucleotide parC sequence in it (Figure IV-4a; we call this “parC- M13” below). For a DNA origami design, as a first trial, we used a pre-existing set of staples that had been originally designed for rectangular origami (twist-corrected, GC-ended). Since the parC sequence is just an addition to the basic scaffold sequence with little (~60 nucleotides) extra difference, the staple strands for the rectangle design should still fold the parC-M13 scaffold strand, leaving the parC addition as a single-stranded loop. Sequence comparison between the regular M13 and the parC-M13 revealed where the loop would fall (Figure IV-5a). We also added a complementary sequence to the parC sequence to make it double-stranded (as is in a plasmid).
Figure IV-4. Design of DNA origami initially proposed for this study. (a) Custom-engineered M13 scaffold for origami with a parC sequence insert. (b) Origami rectangle design that can be tuned to control the distance and angle between the ends of parC strands. (c) Proposed idea of using DNA origami to study the orientation of a ParM filament relative to a ParR/parC complex. (d) Proposed idea of using DNA origami to study the polarity of a ParR/parC complex, which may lead to an expanding origami structure.
m13 parC insert
(169 nt) polarized helix
(based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i x
y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry ofparC-ParR binding to ParM filaments.
10
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i x
y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry ofparC-ParR binding to ParM filaments.
10
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry ofparC-ParR binding to ParM filaments.
10
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry ofparC-ParR binding to ParM filaments.
10
polarized helix (based on parC)
25 nm
+/- might give two independent filaments or no stable filaments.
+/+ might give a single filament symmetric helix
(based on ParR dimer symmetry)
d e
f
g a
b
c
h
i x y
θ
δ δ
parallel binding perpendicular binding
j
k
+/+ weak bond might allow expansion
l m n
Models & hypotheses Experimental platform
3 ways to draw ParM filament
2 models of ParM binding to parC-parR
Using two origami, all relative polarities and two distances can be tested.
Longer origami can be used to test larger separations.
~100 nm x 70 nm, rectangular DNA origami
50 nm +/- 150 nm +/-
50 nm +/+ 150 nm +/+
500 nm +/-
parallel orientation + end attachment
parallel orientation + sidewall attachment
no orientation + sidewall attachment perpendicular orientation
+ end attachment
addition of orienting guide walls no orientation + end attachment
perpendicular orientation + end attachment hypothesis:
hypothesis:
control of parC-ParR orientation (dimers omitted for clarity)
Testing parC-ParR polarity and separation effects.
binding surface for connecting origami via stacking bonds fiduciary mark for orientation
+ -
2 models of helical parC-ParR complex with and promoter omitted for clarity.)
Testing and controlling parC-ParR orientation effects.
ParR
Figure 4: Characterizing the geometry ofparC-ParR binding to ParM filaments.
10
(a) (b)
(c) (d)
85
Figure IV-5. Design of DNA origami used for this study. (a) The design of rectangle origami, used for studies with single-parC origami, which shows the location of the parC sequence (purple) and the hairpin label for contrast in AFM images (“L” mark). (b) Schematic diagrams of the anchors on triangle and rectangle origami for parC strands, used for studies with multiple-parC origami. Each staple strand was extended with T20 at the 5’-end. (c) The design of parC strands. The 5’-end of one of the parC strands (top) was extended by A20, which was bound to a 5’-end T20 extension of staple strands on origami. The 5’-end of the bottom strand was extended by (AAG)5 for fluorescence labeling.
Using this DNA origami structure containing a single parC sequence, we tried to first observe the binding of ParR proteins onto their binding sites of parC on origami, then the binding of ParM filaments to the ParR/parC complexes that would have formed on origami. We first chose to use AFM as our primary measurement tool. Besides being most familiar and available to us, AFM is an ideal instrument for obtaining the details of structures and potentially some interactions at the nanometer scale. We will present AFM results where we could see some interesting behaviors of ParR, depending on its concentration. However, we could also see some limitation of the approach, especially the difficulty in observing ParM, the filament-forming protein, under AFM.
For later investigations, we decided to use other instruments such as gel electrophoresis and fluorescence microscopy as well, which would be more compatible with DNA origami structures containing more than one parC strands. Thus we designed DNA origami that contains multiple parC strands in each origami structure. First, separate parC strands were designed (shown in Figure IV-5c). For incorporation of multiple parC strands onto origami, pre-existing DNA origami designs, such as a triangle and a rectangle, were modified such that each staple strand has a 5’-end extension of 20 T’s (Figure IV-5b), to which parC sequences were designed to link (with an extension of 20 A’s). The top strand of the double-stranded parC was extended at the 5’ end with an
parC-complement, 169nt
A20 (CTT)5-cy3
(AAG)5 parC, 169nt
cy3-streptavidin or
biotin
T20 T20
(a) (b)
(c)
86
overhang of 20 A’s that can bind to the 20-T anchors on origami. In addition to the 169-base sequence that is complementary to the corresponding sequence in the top strand, the bottom strand had (AAG)5-biotin extension at its 5’-end, to which we can incorporate either (CTT)5-Cy3 DNA strand or Cy3-streptavidin for fluorescent labeling.Using the origami with multiple parC strands, we obtained AFM movies of progressive ParR binding onto parC sites on origami, and gel electrophoresis data that show concentration-dependent binding of ParR to parC strands both on origami and free in solution. We also present fluorescence microscope data in a later part of this chapter.