Plenary lecture given by Prof. Katsuhiko Ariga (WPI-MANA, NIMS and University of Tokyo, Japan) on September 12, 2017 in Gramado (Brazil) during the XVI B-MRS Meeting.

1. 1
Handling Molecular Machines by
our Hands
beyond Nobel Prize and
Nanotechnology
Katsuhiko Ariga
WPI-MANA, National Institute for Materials Science (NIMS) &
Graduate School of Frontier Sciences,
The University of Tokyo

2. Thank you very much
for kind invitation

3. I am very happy.

4. Anyway ……

5. Molecular
Machines
Got
Nobel Prize!

6. Dr. Sauvage
Dr. Stoddart
Dr. Feringa

7. Molecular Machine
Manipulation
by
Nanotechnology

8. Dr. Hill Nano Lett. 15, 4793 (2015).
Top-Level Nanotechnology with Future Success
Current-driven Supramolecular Motor with In-situ Surface Chiral Directionality Switching

9. "Nanocar Race"
Car race that use a single molecule as a car

10. Drivers gear up for world’s
first nanocar race
Nature, 544, 278 (2017)

11. 6 Teams from 6 Countries
Sponsor:
PSA Peugeot Citroen
Sponsor:
Swiss Nanoscience Institute
Sponsor: TOYOTA
Sponsor: Volkswagen
Die Gläserne Manufaktur
Car Car
Car
Rotor
Rotor
???

12. NIMS-MANA nanocar
Conformation-Car
O
O
O
O
2.1 nm
0.93
nm
0.81
nm
0.28 nm

13. 1 nm
That's one small step for a molecule, one giant leap for mankind.

14. Race for JAPAN Team
1st DAY
11:00 Race started
11:10 First pulse, 1 nm run
11:15 Software crash
Destroy of Tip, Race tracks, and Nanocars
11:30 Stop of the race
13:00 Recovery of the software and
restart of the race
...Trials to make the Flat nanocars and re-shape
of the tip day and night...
2nd DAY
6:30 Software crash AGAIN, JAPAN Team
retired.

15. Race Records
~1000 nm?

16. Working Day and Night
Fair Play Prize for
NIMS-MANA JAPAN Team

17. Beyond
Nobel Prize &
Nanotechnology

18. Freely tune molecules
just by our hands
Hand-Operating Nanotechnology
Beyond
Nobel Prize &
Nanotechnology
More general & versatile

19. Necessary forces to
control molecules

20. Necessary forces to control molecules
1 10 100 1000 10000
pN
Chemical bond:
C-O (calcd; 4300 pN) ref1
C-C (calcd; 4120 pN) ref1
C-N (calcd; 4100 pN) ref1
C-Si (2000 pN) ref1
covalent bond in carbonic
anhydrase B unfolding (1700 pN)
ref 2
S-Au (1400 pN) ref 1
Weak covalent
bond:
quadruple H-bonded ureido-
4[1H]-pyrimidinone (UPy) system
(148 pN) ref3
C-O carboxyl groups (in vaccum,
Hex, 1-PrOH, clcd; 148, 144, 71,
57 pN) ref 4
O-H carboxyl groups (in water,
clcd; 17 pN) ref 4
Strong protein
interactions:
Antigen/Antibody (224, 160, 120,
55 pN) ref 5, 7, 9, 17
Biotin/Streptavidin (160, 150, 55
pN) ref6, 8, 16
Biotin/Avidin (130, 50 pN) ref8,
18
PDZ domain/recognition peptide
(120 pN) ref 10
P-selectin/PSGL-1 ligand (115
pN) ref 11
p53/mdm2 (105 pN) ref 12
DNA assembly:
Melting of dsDNA into single strands
(150 pN) ref 37
B to S form (65 pN) ref 38
phi DNA packing motor (57 pN) ref
39
Adenine/Thymine (54 pN) ref 40
RNA polymerase stalling (25 pN) ref
41
Unzipping of poly(dG-dC),
inhomogeneous, poly(dA-dT) DNA
(20, 12, 9 pN) ref 37, 42
Straighten (6 pN) ref 43
Conformational
change of
protein:
Stretching alpha-helix (200 pN)
ref44
Unfolding of ubiquitin (160 pN) ref
45
Unfolding of fibrinogen (94 pN) ref
94
Small force:
Force to produce energy 1kT by
pull of 1nm at 300K (4 pN) ref
47
Force by membrane potential
per elementary charge in protein
pore (3 pN) ref48
Moderate protein
interactions:
Azurin/cytocrom c551 (95 pN) ref 13
p53/azurin (75 pN) ref 14
Carbonic anhydrase/inhibitor (65 pN) ref 15
Alpha-beta integrin/GRGDSP (20 pN) ref 19
Thrombin/aptamer (5 pN) ref 20
Unfolding of alpha-helical protein (50 pN)
ref 29
Unfolding of a single Ankyrin repeat (37 pN)
ref 26
Cadherin-Cadherin complex (35 pN) ref 30
Refolding single Ankyrin repeat (32 pN) ref
26
Unfolding of myosin II (31 pN) ref 31
Weak protein
interactions:
gating spring of channel (10 pN)
ref 32
Kinesin stall force on
microtubule (7 pN) ref 33
Focal adhesion and
cytoskeleton (1 pN) ref 34
cell-cell contacts (1 pN) ref 35
Force detectable by hair cell
bundle (1 pN) ref 36
Myosin V
3 pN
RNA polymerase
23 pN
azobenzene
1000 pN
N N
N O
spiropyran
2000 pN
References from Anishkin et al. PNAS, 2014, 111, 7899 and others.

21. How to control a molecule:
Unexplored region
1 10 100 1000 10000
pNMyosin V
3 pN
RNA polymerase
23 pN
azobenzene
1000 pN
N N
N O
spiropyran
2000 pN
Not well-controlled by external forces
DNA RNA unzipping
0.5-15 pN
F0F1-ATPase
8 pN
Conformation/
Interactions/
Soft Bio-Control
Photo isomerization/
Covalent bond formation
Force
Mechano-chemistry
Conventional
operations

22. Small Forces (pN) for Bio-functions
D. G. Rodriguez et al.
Science, 2012, 338, 910.
D. E. Discher et al.
Science, 2005, 318, 1139.
M. P. Sheetz et al.
Science, 2009, 323, 638.
Cell migration (infiltration of cancer)
Cell differentiation (ES/iPS)
Protein conformational change (change in ligand binding and enzymatic activity)
…. artificial controls under challenges

23. Molecule/Nano Macroscopic
Materials ScienceNanotechnology
Size-Increase (Assembly)
Reduce Dimension: 3D to 2D
Macroscopic
Molecule/Nano
Nanotechnology & Materials Science

24. Surface pressure (F): 1-70 mN/m
Estimated force per molecule
≅ 0.5-35 pN
Estimated pressure
≅ 0.5-35 MPa
Estimated energy
(integral of π-A curve)
≅ 1 kcal/mol
Thermal fluctuation occurs when energy
barrier is under 20 kcal/mol.
Oki, M. Proc. Jpn. Acad., Ser. B 2010, 86,
867–883.
Force/Energy at the Air-Water Interface: Langmuir Monolayer System
0
10
20
30
40
50
0.3 0.6 0.9 1.2 1.5 1.8
Surfacepressure[mNm–1]
Molecular area [nm2]
Eintegral=~1 kcal mol-1
30
1
2
0
1
0
Mechanical
Energy
Eintegral

25. Langmuir technique can cover
unexplored region
1 10 100 1000 10000
pNMyosin V
3 pN
RNA polymerase
23 pN
azobenzene
1000 pN
N N
N O
spiropyran
2000 pN
Well-controlled at the air-water interface
DNA RNA unzipping
0.5-15 pN
F0F1-ATPase
8 pN
Conformation/
interactions
Photo isomerization/
Covalent bond formation
Mechano-chemistry

26. Breaking Common Sense to Create A New Road
First Demonstration
Catch & Release
A Molecule
by Our Hand Motions

27. Molecular Machine at Dynamic Interface
Macroscopic Dimension
Monolayer at Dynamic Interface
Molecular-level Dimension
Environment with both molecular
and macroscopic characteristics!
Dynamic but Flat
Invisible molecular
machines
Active Film
Molecular machines
have got Nobel Prize!
Useless ???
Nanocar under vacuum

28. 28

29. 29

30. 30

31. 31

32. 32
Bulk Operation
Access to
Molecular World
Connection between
molecular (nano) world
and real (visible) world
Hand-Operating Nanotechnology
Molecular Machine at Interface

33. Breaking Common Sense to Create A New Road
Second Demonstration
Tuning of Receptors
New Mode of Molecular Recognition
Tuning Receptor

34. Second Demonstration
Finer Tuning of Receptors
Precise Discrimination
of Biomolecules
by Mechanical Motions
New Mode of Molecular Recognition
Tuning Receptor

35. J. Am. Chem. Soc., 128, 14478 (2006).
Phys. Chem. Chem. Phys., 13, 4895 (2011).
Hand-Operating Nanotechnology
Chiral Resolution by Hand Motion
First Achievement Since Dr. Pasteur

36. J. Am. Chem. Soc., 132, 12868 (2010).
Surface Pressure / mNm-1
BindingConstant(KU/KT)
[LiCl] = 0 mM
[LiCl] = 10 mM
Best Condition

37. Traditional Host-Guest Systems
Simple Use of Energy Minimum
Mechanism for Most of Molecular Machines
(2nd Nobel prize)
Continuous Modulation to Find
Best Solution from Numerous Candidates
Mechanical Tuning of
Conformation of Host Molecule
Energy
Energy Hand-Operation
One State
Tuning
Use soft materials softly.
Pioneer: S. Shinkai et al., Tetrahedron Lett. (1979),
Chem. Lett. (1980), Chem. Commun. (1980), JACS (1981)
Switching
Switching between
Separate States
Energy
Origin of Supramolecular Chemistry
(1st Nobel prize)

38. New concept may come
Functional Conformer Science
Tuning molecules toward best function
at outside of simple equilibrium
All possible conformers
Unexplored functions
Huge possibilities
So far, we only investigated
most stable and most probable state

39. Breaking Common Sense to Create A New Road
How is energy efficiency of
Hand-Manipulation of Molecule
at interface?
Exploration with model system

40. 0
10
20
30
40
50
60
70
80
90
100
Energy efficiency:
High efficiency with simple direct contact
of force and motion.
Can we extend this assumption to molecular-level?
Molecular-
Level
?

41. Simple mechanical molecular machine
Hydrophobic
alkyl chain
Hydrophilic
polyether chain
OO
O
O
O
O
O
O
Dr. Waka Nakanishi

42. Recent Work:
Mechanochemical Control of
Simple Molecular Machine, a Nano-Pliers
Compression
Expansion
Angew. Chem., Int. Ed., 54, 8988 (2015).
Hydrophobic
alkyl chain
Hydrophilic
polyether chain

43. Theory & Experiments
Revealed Motion of a Molecule
Hydrophobic
alkyl chain
Hydrophilic
polyether chain
1 mN/m
10 mN/m
20 mN/m
30 mN/m
2.0
1.0
0
−
1.0
−
2.0
200 250 300 350
λ /nm
θA/mdegnm2
3.0
−
3.0
Force
200 250 300 350
λ /nm
600
400
200
0
−
200
−
400
−
600
−
800
800 −
90°
∆ε/M-1cm-1
−
80°−
70°−
60°−
50°
Close of pliers
Experimental data from transferred monolayer Simulations of single molecule
TDDFT(B3LYP/6-31Gdp)

44. MD Simulations
Revealed Structures of Molecules in an Assembly
Expanded Compressed
Dr. D. Cheung, National University of Ireland Galway
Hydrophobic
alkyl chain
Hydrophilic
polyether chain

45. Energy given at the air-water interface
0.5 1
10
20
30
40
0
Molecular Area / nm
2
SurfacePressure/mNm
-1
Energy by
Thermodynamics

46. Characteristic Properties at the Air-Water Interface 3
Estimated φ and obtained
torsional energies
v.s. applied mechanical
force
Control of conformational change
of molecules and proteins is
possible.
Efficient energy conversion is
expected.
molecule

47. 0
10
20
30
40
50
60
70
80
90
100
Electricity generation Machines at macro size Machines at nano size
Mechanical molecular machine works
with high efficiency.
N N
Direct mechanical driving is best method.

48. Unified Understanding
on
Relation between
Force and Motion
from MACRO to NANO

49. Comparison of Forces [N]
Myosin V
3 pN
RNA polymerase
23 pN
Macroscopic Molecular Machine
Car
45 kN
Biology
Size 1 m 10 nm 1 nm
ac
F0F1-ATPase
8 pN
Binaphthyl
1 pN
Binaphthyl
+Lipid Matrix
1 pN
Interfacial Mechanical Molecular Machine
Human
8 kN
Beetle
10 N

50. Weight/Force x Length [g/Nm]
Operated weight per energy
Traditional
Molecular Machines
Interfacial Molecular Machines
Biology
Macroscopic
Machines
1000
100
10
1
0.1
0.01
0.001
0.0001
[g/Nm]
nm µm mm m
Size
Car
Human
Beetle
ATPase
Myosin V
RNA Polymerase
Stilbene
Spiropyran

51. Breaking Common Sense to Create A New Road
Next Challenges
Control of Life
by
Hand-Operating Nanotechnology

52. DNA Origami
Dr. Yonamine
Collaboration with Prof. Murata (Tohoku Univ.)

53. DNA Origami
fold
M. Endo et al. Biomater. Sci., 1, 347 (2013)

54. Shape-forming and assembly
of DNA origami
is pre-determined (programed)
with DNA sequence.
Concept of DNA Origami
We want to control them
by our hands beyond
program.

55. 7,249 base
Cationic lipid
Lipid-modified DNA origami sheet
90 nm
65nm
DNA origami sheet
ss DNA
M13 mp18 Staple DNA strands
Langmuir Film of Lipid-modified DNA Origami Sheet
1-D fusion
Air-water
interface
Langmuir film
Compression
Expansion

56. Langmuir-Blodgett Film of DNA Origami

57. Lipid (2C18N+)
Lipid-modified
DNA sheet
π-A isotherm and AFM Image of Lipid-DNA Origami Sheet
Lipid-modified
DNA origami sheet
DNA origami sheet
1.0 nm1.5 nm
200 nm 200 nm
32 mN/m
First Example of DNA Origami LB Film

58. Repeat Compression and Expansion (3 ⇄ 30 mN/m)
Air-water
interface
Langmuir film
Compression
Expansion
?
Transfer at
32 mN/m

59. 1000 nm
0 repeat
Pressure[mN/m] 0
3
30
Time
32

60. 1 repeat
1000 nm
Pressure[mN/m] 1
3
30
Time
32

61. Belt-shape fusion
2 repeat
1000 nm
Pressure[mN/m]
3
30
Time
2
32

62. 0 nm
93 nm
68 nm
74 nm
1740 nm
H L
W
L / H
Aspectratio
W / H
1500
500
1000
0
0 2
Number of cycle
0 2
20
10
0
30
L / W
0 2
500 nm
0 repeat 2 repeat
500 nm
Supramolecular Polymerization
of DNA Origami
Phys. Chem. Chem. Phys. 2016, 18, 12576–12581.

63. Cell Differentiation
Cell Fate Control at Interfaces.

64. Fluidity of Microenvironment
64Uto, K. & Ebara M. et al. ACS Biomater. Sci. Eng. 2016, 2, 446–453.
Fluid for cellular microenvironment

65. Stiffness of Biological Tissues
65Engler A. J. et al. Cell 2006, 126, 677–689.
Super-Hard
Super-Soft

66. Super Hard! Fullerene Crystals
66
• 1D fullerene crystal
• Uniform structure with high aspect ratio
– ca. 500 nm in diameter
– > 100 µm in length
• Biocompatibility
– Phagocytosis by macrophage-like cell
– Low cytotoxicity
1 µm
Adv. Biomed. Res. Proceedings 2010, 89.
J. Mater. Res. 2002, 17, 83.
whisker sheet
Fullerene Whisker / Nanowhisker
Liquid–Liquid Interface Precipitation (LLIP) method
Crystals grow at
liquid–liquid interface.
• Various structure

67. Alignment of Fullerene Whiskers
67
FW film
Compress Transfer Dry
Aligned FW film
Aligned FW
2.5 µm20 µm 500 nm
Optical microscopy SEM Atomic force microscopy
Langmuir–Blodgett (LB) approach
Adv. Mater. 2015, 27, 4020–4026.
Dr. Minami
Mr. Kasuya

68. Elongation of Cell Morphology by FW Pattern
68
Bare glass Random FW Aligned FW
Fluorescent analysis of cell morphology Stain: Cytoskeleton/Nuclei
100 µm
Aspect ratio of cells Myoblast
Elongated shape
Early stage
of myogenesis
Aligned FW induced early stage of myogenesis,
and controlled the growth direction of myoblasts.
2.3
4.0
Adv. Mater. 2015, 27, 4020–4026.

69. Promotion of Differentiation
69
Myogenesis under low serum environment
Aligned FWBare glass
Gene expression
Fusion index
Cell adhesion Morphology Differentiation
Elongation Fusion
Aligned FW induced myogenic differentiation.
Stain: Myosin heavy chain/Nuclei
Adv. Mater. 2015, 27, 4020–4026.

70. Super Soft! Liquid–Liquid Interfaces
70
Liquid–Liquid interfaces
• Fluid environment
Perfluorocarbons
• Fully fluorinated substances
• Immiscible with water as well as
common organic solvents
• Heavier density than water
Perfluorocarbon
Water
From wikipedia
RfOct RfMCH RfPh
Dr. Minami

71. Spreading and Morphological Investigations
71
Cells at RfOct interface only showed spreading morphology.
ACS-AMI Accepted
Spreading

72. Suppression of Differentiation
72
Myogenic differentiation can be suppressed by fluid microenvironment.

73. Can interfaces determine cell fate?
73
Stemness maintenance
Cell Death Differentiation 2010, 17, 1230–1237.
Unstable
iPS cells
• iPS cells have pluripotency.
– Easy to differentiate
– Necessary to maintain their pluripotency
• Stemness maintenance
Interfacial culture

74. How to control molecule, systems, & life
1 10 100 1000 10000
pN
Myosin V
3 pN
RNA polymerase
23 pN azobenzene
1000 pN
N N
N O
spiropyran
2000 pN
Not well-controlled by external forces
New Challenge
DNA RNA unzipping
0.5-15 pN
F0F1-ATPase
8 pN
Conformation/
Interactions/
Soft Bio-Control
Photo isomerization/
Covalent bond formation
Force
Mechano-chemistry
Conventional
operations

75. Write
Delete
Rewrite
C60 molecule
Bit density: 200 Tb/in2
Monomolecular level ultrahigh density memory
Atomic Switch
High-Tech-Driven Nanotechnology
Hand-Operating
Nanotechnology
Easy-Action-Based Nanotechnology
Nanotechnology for
Common Use in
Daily Life
However ………….
They are only operations
under selected conditions
with specialized equipment

76. Thank you very much
Members
Antonio Robson
Andrei