This paper reports a method for trapping circular DNA molecules and imaging the dynamics with high spatial resolution using a micropillar-array device. We successfully trapped circular bacterial artificial chromosome DNA molecules at a micropillar-based “ring toss” in the laminar flow of a microchannel under a fluorescence microscope and demonstrated the imaging of their extension by flow and condensation process induced by spermine solution. DNA molecules were visualized in an extended loop conformation, allowing high spatial resolution, and the results showed that the dynamics is induced by the microfluidic control of the surrounding chemical environment. The method is expected to lead to the elucidation of the physical characteristics and the dynamics of circular DNA molecules.

1.
H.
Kabata
,
O.
Kurosawa
,
I.
Arai
,
M.
Washizu
,
S.
Margarson
,
R.
Glass
, and
N.
Shimamoto
, “
Visualization of single molecules of RNA polymerase sliding along DNA
,”
Science
262
,
1561
1563
(
1993
).
2.
Z.
Gueroui
,
C.
Place
,
E.
Freyssingeas
, and
B.
Berge
, “
Observation by fluorescence microscopy of transcription on single combed DNA
,”
Proc. Natl. Acad. Sci. U.S.A.
99
(
9
),
6005
6010
(
2002
).
3.
N. A.
Tanner
,
J. J.
Loparo
,
S. M.
Hamdan
,
S.
Jergic
,
N. E.
Dixon
, and
A. M.
van Oijen
, “
Real-time single-molecule observation of rolling-circle DNA replication
,”
Nucleic Acids Res.
37
,
e27
(
2009
).
4.
M.
Levy-Sakin
,
A.
Grunwald
,
S.
Kim
,
N. R.
Gassman
,
A.
Gottfried
,
J.
Antelman
,
Y.
Kim
,
S. O.
Ho
,
R.
Samuel
,
X.
Michalet
,
R. R.
Lin
,
T.
Dertinger
,
A. S.
Kim
,
S.
Chung
,
R. A.
Colyer
,
E.
Weinhold
,
S.
Weiss
, and
Y.
Ebenstein
, “
Toward single-molecule optical mapping of the epigenome
,”
ACS Nano
8
,
14
26
(
2014
).
5.
B. R.
Cipriany
,
R.
Zhao
,
P. J.
Murphy
,
S. L.
Levy
,
C. P.
Tan
,
H. G.
Craighead
, and
P. D.
Soloway
, “
Single molecule epigenetic analysis in a nanofluidic channel
,”
Anal. Chem.
82
,
2480
2487
(
2010
).
6.
P. R.
Bianco
,
L. R.
Brewer
,
M.
Corzett
,
R.
Balhorn
,
Y.
Yeh
,
S. C.
Kowalczykowski
, and
R. J.
Baskin
, “
Processive translocation and DNA unwinding by individual RecBCD enzyme molecules
,”
Nature
409
,
374
378
(
2001
).
7.
R.
Galletto
,
I.
Amitani
,
R. J.
Baskin
, and
S. C.
Kowalczykowski
, “
Direct observation of individual RecA filaments assembling on single DNA molecules
,”
Nature
443
,
875
878
(
2006
).
8.
E.
Raderschall
,
E. I.
Golub
, and
T.
Haaf
, “
Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage
,”
Proc. Natl. Acad. Sci. U.S.A.
96
,
1921
1926
(
1999
).
9.
T.
Strick
,
J.
Allemand
,
V.
Croquette
, and
D.
Bensimon
, “
Twisting and stretching single DNA molecules
,”
Prog. Biophys. Mol. Biol.
74
,
115
140
(
2000
).
10.
E. V.
Volpi
and
J. M.
Bridger
, “
FISH glossary: An overview of the fluorescence in situ hybridization technique
,”
BioTechniques
45
,
385
409
(
2008
).
11.
H.
Barseghyan
,
W.
Tang
,
R. T.
Wang
,
M.
Almalvez
,
E.
Segura
,
M. S.
Bramble
,
A.
Lipson
,
E. D.
Douine
,
H.
Lee
,
E. C.
Délot
,
S. F.
Nelson
, and
E.
Vilain
, “
Next-generation mapping: A novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis
,”
Genome Med.
9
,
90
(
2017
).
12.
K.
Terao
,
M.
Washizu
, and
H.
Oana
, “
On-site manipulation of single chromosomal DNA molecules by using optically driven microstructures
,”
Lab Chip
8
,
1280
1284
(
2008
).
13.
K.
Terao
,
C.
Masuda
,
R.
Inukai
,
M.
Gel
,
H.
Oana
,
M.
Washizu
,
T.
Suzuki
,
H.
Takao
,
F.
Shimokawa
, and
F.
Oohira
, “
Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope
,”
IET Nanobiotechnol.
10
,
124
128
(
2016
).
14.
H.
Oana
,
K.
Nishikawa
,
H.
Matsuhara
,
A.
Yamamoto
,
T. G.
Yamamoto
,
T.
Haraguchi
,
Y.
Hiraoka
, and
M.
Washizua
, “
Correction: Non-destructive handling of individual chromatin fibers isolated from single cells in a microfluidic device utilizing an optically driven microtool
,”
Lab Chip
14
,
696
704
(
2014
).
15.
J. R.
Moffitt
,
Y. R.
Chemla
,
S. B.
Smith
, and
C.
Bustamante
, “
Recent advances in optical tweezers
,”
Annu. Rev. Biochem.
77
,
205
228
(
2008
).
16.
K.
Hirano
,
H.
Nagata
,
T.
Ishido
,
Y.
Tanaka
,
Y.
Baba
, and
M.
Ishikawa
, “
Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping
,”
Anal. Chem.
80
,
5197
5202
(
2008
).
17.
Y.
Matsuzawa
,
K.
Hirano
,
K.
Mori
,
S.
Katsura
,
K.
Yoshikawa
, and
A.
Mizuno
, “
Laser trapping on an individual DNA molecule folded using various condensing agents
,”
J. Am. Chem. Soc.
121
,
11581
11582
(
1999
).
18.
K.
Hirano
and
Y.
Baba
, “
Manipulation of single coiled DNA molecules by laser clustering of microparticles
,”
Appl. Phys. Lett.
80
,
515
517
(
2002
).
19.
I. D.
Vlaminck
and
C.
Dekker
, “
Recent advances in magnetic tweezers
,”
Annu. Rev. Biophys.
41
,
453
472
(
2012
).
20.
M.
Kumemura
,
D.
Collard
,
N.
Sakaki
,
C.
Yamahata
,
M.
Hosogi
,
G.
Hashiguchi
, and
H.
Fujita
, “
Single-DNA-molecule trapping with silicon nanotweezers using pulsed dielectrophoresis
,”
J. Micromech. Microeng.
21
,
045026
(
2011
).
21.
J. O.
Tegenfeldt
,
C.
Prinz
,
H.
Cao
,
S.
Chou
,
W. W.
Reisner
,
R.
Riehn
,
Y. M.
Wang
,
E. C.
Cox
,
J. C.
Sturm
,
P.
Silberzan
, and
R. H.
Austin
, “
The dynamics of genomic-length DNA molecules in 100-nm channels
,”
Proc. Natl. Acad. Sci. U.S.A.
101
,
10979
10983
(
2004
).
22.
E. T.
Lam
,
A.
Hastie
,
C.
Lin
,
D.
Ehrlich
,
S. K.
Das
,
M. D.
Austin
,
P.
Deshpande
,
H.
Cao
,
N.
Nagarajan
,
M.
Xiao
, and
P. K.
Kwok
, “
Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly
,”
Nat. Biotechnol.
30
,
771
776
(
2012
).
23.
K.
Terao
,
H.
Kabata
, and
M.
Washizu
, “
Extending chromosomal DNA in microstructures using electroosmotic flow
,”
J. Phys. Condens. Matter
18
,
S653
S663
(
2006
).
24.
T.
Yasui
,
N.
Kaji
,
M. R.
Mohamadi
,
Y.
Okamoto
,
M.
Tokeshi
,
Y.
Horiike
, and
Y.
Baba
, “
Electroosmotic flow in microchannels with nanostructures
,”
ACS Nano
5
,
7775
7780
(
2011
).
25.
M.
Washizu
and
O.
Kurosawa
, “
Electrostatic manipulation of DNA in microfabricated structures
,”
IEEE Trans. Ind. Appl.
26
,
1165
1172
(
1990
).
26.
K.
Hirano
,
Y.
Matsuzawa
,
H.
Yasuda
,
S.
Katsura
, and
A.
Mizuno
, “
Orientation control, cutting in order, and recovery of single DNA molecules using the electric effect inside channels on a glass substrate
,”
J. Cap. Elec. Microchip Tech.
6
,
13
18
(
1999
).
27.
M.
Alizadehheidari
,
E.
Werner
,
C.
Noble
,
M.
Reiter-Schad
,
L. K.
Nyberg
,
J.
Fritzsche
,
B.
Mehlig
,
J. O.
Tegenfeldt
,
T.
Ambjörnsson
,
F.
Persson
, and
F.
Westerlund
, “
Nanoconfined circular and linear DNA: Equilibrium conformations and unfolding kinetics
,”
Macromolecules
48
,
871
878
(
2015
).
28.
R.
Schleif
, “
DNA looping
,”
Annu. Rev. Biochem.
61
,
199
223
(
1992
).
29.
O. L.
Kantidze
and
S. V.
Razin
, “
Chromatin loops, illegitimate recombination, and genome evolution
,”
BioEssays
31
,
278
286
(
2009
).
30.
L. F.
Liu
and
J. C.
Wang
, “
Supercoiling of the DNA template during transcription
,”
Proc. Natl. Acad. Sci. U.S.A.
84
,
7024
7027
(
1987
).
31.
J. C.
Wang
, “
DNA topoisomerases
,”
Annu. Rev. Biochem.
65
,
635
692
(
1996
).
32.
Y.
Pommier
,
E.
Leo
,
H.
Zhang
, and
C.
Marchand
, “
DNA topoisomerases and their poisoning by anticancer and antibacterial drugs
,”
Chem. Biol.
17
,
421
433
(
2010
).
33.
L. F.
Liu
, “
DNA topoisomerase poisons as antitumor drugs
,”
Annu. Rev. Biochem.
58
,
351
375
(
1989
).
34.
I. F.
King
,
C. N.
Yandava
,
A. M.
Mabb
,
J. S.
Hsiao
,
H. S.
Huang
,
B. L.
Pearson
,
J. M.
Calabrese
,
J.
Starmer
,
J. S.
Parker
,
T.
Magnuson
,
S. J.
Chamberlain
,
B. D.
Philpot
, and
M. J.
Zylka
, “
Topoisomerases facilitate transcription of long genes linked to autism
,”
Nature
501
,
58
62
(
2013
).
35.
L.
Vokalov
,
J.
Durdiakova
, and
D.
Ostatnikova
, “
Topoisomerases interlink genetic network underlying autism
,”
Int. J. Dev. Neurosci.
47
,
361
368
(
2015
).
36.
T. T.
Perkins
,
D. E.
Smith
,
R. G.
Larson
, and
S.
Chu
, “
Stretching of a single tethered polymer in a uniform flow
,”
Science
268
,
83
87
(
1995
).
37.
T. T.
Perkins
,
D. E.
Smith
, and
S.
Chu
, “
Single polymer dynamics in an elongational flow
,”
Science
276
,
2016
2021
(
1997
).
38.
K.
Gunther
,
M.
Mertig
, and
R.
Seidel
, “
Mechanical and structural properties of YOYO-1 complexed DNA
,”
Nucleic Acids Res.
38
,
6526
6532
(
2010
).
39.
C. U.
Murade
,
V.
Subramaniam
,
C.
Otto
, and
M. L.
Bennink
, “
Force spectroscopy and fluorescence microscopy of dsDNA–YOYO-1 complexes: Implications for the structure of dsDNA in the overstretching region
,”
Nucleic Acids Res.
38
,
3423
3431
(
2010
).
40.
C. W.
Tabor
and
H.
Tabor
, “
Polyamines
,”
Annu. Rev. Biochem.
53
,
749
790
(
1983
).
41.
V. A.
Bloomfield
, “
Condensation of DNA by multivalent cations: Considerations on mechanism
,”
Biopolymers
31
,
1471
1481
(
1991
).
42.
M.
Takahashi
,
K.
Yoshikawa
,
V.
Vasilevskaya
, and
R.
Khokhlov
, “
Discrete coil−globule transition of single duplex DNAs induced by polyamines
,”
J. Phys. Chem. B
101
,
9396
9940
(
1997
).
43.
A.
Tsuji
and
K.
Yoshikawa
, “
ON−OFF switching of transcriptional activity of large DNA through a conformational transition in cooperation with phospholipid membrane
,”
J. Am. Chem. Soc.
132
,
12464
12471
(
2010
).
44.
K.
Hirano
,
M.
Ichikawa
,
T.
Ishido
,
M.
Ishikawa
,
Y.
Baba
, and
K.
Yoshikawa
, “
How environmental solution conditions determine the compaction velocity of single DNA molecules
,”
Nucleic Acids Res.
40
,
284
289
(
2012
).
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