Hiroshi Sugiyama Department of Chemistry, Graduate School of Science Institute for Integrated Cell-Material Sciences (iCeMS)

Kyoto University1

100 nm

生体分子機能論 2018-4３次元構造２

Advanced Course in Molecular Biology and Biochemistry

2

Haloarcula japonica strain TR-1

高度好塩菌とは｢高い塩濃度の環境下でしか生きられない｣細菌のことです。どれぐらい高い濃度かというと、なんと20～25％、ほとんど飽和食塩水に近い環境です。海水でもせいぜい2～3％ですから、いかにものすごい環境であることがおわかり頂けると

思います。地球上にはこのような環境が、イスラエルの死海のような塩湖、南米高地の塩平原や、塩

田等にあり、これら好塩菌もこのようなところで見出されます。たまに塩漬けにした魚等で生育し、 この魚等を真っ赤に染めてしまったりもします。

FRET ( Föster resonance energy transfer )

Å

R0 : Förester critical distance

JDA : doner/acceptor spectral overlap n : reflective index of medium

φD : quantum yield of donor

e1, e2 : the unit vectors of the donor and acceptor transition dipoles

e12 : the unit vector between their centers

Donor Acceptor

1

450 mM NaCl

Basics1 Basic elements of nucleic acids and their synthesis

2 Sequencing of DNA

3 3D structure of DNA 1

4 3D structure of DNA 2

Chemistry 5) DNA alkylation

6) Hydrogen abstraction 1

7) Hydrogen abstraction 2

8) Charge transfer

Biology 9) Epigenetics 1

10) Epigenetics 2

11) ATRX

3D structure of DNA 2

1) syn vs anti

2) Parallel and antiparallel

G-quadruplex

G

G

G

G

K+

Hybrid structure or 3 +1 structure

Bioorg.Med.Chem. 2006,14, 5584.AGGG(TTAGGG)3

Telomestatin

Hybrid structure of human telomere G-quadruplex

T-loop formation

Each cell division causes telomere

shortening (100-200bp) and after

30-40 divisions apoptosis occurs.

Most of cancer cells express

telomerase to obtain immortality.

A

T

T

T

A

T

synanti

anti

G

G

GG

G

G

G

G

GG

G

GG

G

G

G

GG

G

G

G

G

G

5' A Diagonal loop

Lateral loop

External TTA loop

3'

3'

5' A

K+ Form Na+ Form

Nature 417, 876-880 (2002) Structure 356, 164-168 (1992)

G

K. Shin-ya et al. J. Am. Chem. Soc. 2001, 123, 1262-1263.

G4結合性リガンドと疾患との関連

テロメスタチン（天然物）

テロメラーゼ活性阻害剤

染色体末端テロメア領域

✓ テロメラーゼ活性阻害

二本鎖領域

新しい疾患治療薬の創生✓ 疾患関連遺伝子の転写阻害

G4を作用機序とする最初の治験薬剤

カルフロキシン

5-アミノレブリン酸

PpIX (or Hemin)

ATRX治療薬候補

5’-G≥3N1-7G≥3N1-7G≥3N1-7G≥3-3’

18

W. G. Rice et al., Cancer Res. 2009, 69, 7653-7661. N. Shioda et al., Nat. Med. 2018, 24, 802 –813.

G4の多様な生物学的機能 ~ G4結合性リガンドの分子プローブとしての応用 ~

クロマチン再構成・複製起点の目印

転写因子結合阻害 ゲノム不安定性

エピジェネティック変化

Replication process

S. Asamitsu, T. Bando, H. Sugiyama, Chem. Eur. J. 2018 (review), in press.

26

ゲノム上の個々のG4を識別可能な分子プローブの必要性が高まっている.

G4抗体によるG4 DNA・RNAの可視化 ~ 生体内のG4形成を示唆 ~ 17

Nat. Chem. 2013, 5, 182-186.

赤 : G4抗体 (BG4)

緑 : TRF2 (テロメア結合タンパク)

Nat. Chem. 2014, 6, 75-80.

T-loop formation

J. Am. Chem. Soc. 128, 9963-70 (2006)

Nucleic Acids Res. 34, 2723-35 (2006)

Bioorg. Med. Chem. 14, 5584-91 (2006)

J. Am. Chem. Soc. 130, 16470 (2008)

syn

G-quadruplex Stabilizes T-Loop Formation

3’-TGGGTTT

5’-GGGTTAGGGTTAGGGTTT

5’-CTGTAGCTCAACATGT-GGAGACTCTAGAGTGTTCCTGATGGCCGTGAA-----TTCAAGGCGGTGGGTGCGCGTTGCTCCTCACT-GAACACCCTGAACAAA-3’

3’-CCTCTGAGATCTCACAAGGACTACCGGCACTT-5’ AAGTTCCGCCACCCACGCGCAACGAGGAGTGA-5’

3’-TGGGTTT-

3’-CTAAGAGAACAAACGA-GCTCGCCTCTGAGATCTCACAAGGACTACCGGCACTT-----AAGTTCCGCCACCCACGCGCAACGAGGAGTGAAGGAG-TACAACAAATAACAGC-5’

5’-CGAGCGGAGACTCTAGAGTGTTCCTGATGGCCGTGAA-3’ TTCAAGGCGGTGGGTGCGCGTTGCTCCTCACTTCCTC-3’

5’-GGGTTAGGGTTAGGGTTT-

64mer dsDNA

74mer dsDNA

In the presence of 100 mM KClIn the absence of KCl

1000 x 750 nm 1000 x 750 nm

[ 3 + 1 ]G-quadruplex

G-quadruplex formation: 44% (220 frames counted)

A B

C D

+ KCl

225 x 225 nm23

G-quadruplex Formation in DNA Frame

20 mM Tris buffer (pH 8.0)10 mM MgCl2

100 mM KCl20 mM Tris buffer (pH 8.0)10 mM MgCl2

10 sec / frame

G-quadruplex formation was directly observed at single molecular level.

200 x 200 nm

A B

C D

+ KClA B

C D

J. Am. Chem. Soc. 132, 16311 (2010)

G-quadruplex Formation in DNA Frame

Angew. Chem. Int. Ed. 2014, 53, 4107 –4112

Quadruplex/Duplex Complex mimicking telomere region

hPIP-cIKP hybrids targeting a quadruplex/duplex complex structure

Collaborative project

with Prof Anh Tuan Phan

Chem. Eur. J. 2018, 24, 4428-4435.

FRET melting-point assays

5’-AGGGTGGGTAGGGTGGGTAAGGTGCG-TAMRA-3’

3’-ATTCCACGC-FAM-5’

Receptor was generated from PDB code: 5DWW and modified. (S. Neidle, G. N. Parkinson et al. J. Am. Chem. Soc., 138, 1226 (2016))

hPIP-cIKP hybrid

Quadruplex-specific binder

Duplex minor groove binderChembiochem. 2016, 17, 1317-22

PIP dimer stabilizes G-quadruplex structure

Biochem. 2018, 57, 498-502.

5’-GCGGTTTAATGTGAAATGGGTGGGTGGGTGGGTAAACTCTTATACGCG-3’

3’-CGCCAAATTACACTTTACCCACCCACCCACCCATTTGAGAATATGCGC-5’

Binding site Binding site

G-quadruplex forming region

PIP monomer (1)PIP dimer (2)

K+

G-quadruplex (GQ) 3’

5’

2.7 nm

Optical tweezers

force

fold

unfold

50 nm

X

laser trap 1 laser trap 2

50 nm

human telomere G-quadruplex: TTAGGGTTAGGGTTAGGGTTAGGGTTA

GQ in DNA nanocage

5-15 nm

Force-extension curve

Forc

e (p

N)

extension (nm)

force

ΔL

relaxing

stretching

Nature Chem. 3, 782 (2011)

G-quadruplex in DNA Nanocage Prof Hanbin Mao (Kent State Univ)

nanocage

Within nanocage

3’

5’

5 X 5 nanocage

1 2 3 4

16

15

14

5

6

7

8

910111213

Stretching and relaxing F-X curves of a GQ hosting sequence inside the nanocage

8 nm8 nm 8 nm

Stability of GQ in the nanocages (38.6 pN) is about twice than that without nanocage (~20 pN).

GQ relaxing

stretching

Without nanocage

3’

5’

Change-in-contour-length (ΔL) Unfolding force histograms

unfold

Nature Nanotechnology, 12, 582 (2017)

G-quadruplex in DNA Nanocage Prof Hanbin Mao (Kent State Univ)

PNAS 115, 9539 (2018)

i Motif in DNA Nanocage Prof Hanbin Mao (Kent State Univ)

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

R% l 5 R%

8A5 u z l * t z 7% %7 u l

% u p l 6

R% * 8A5 k z y k

l z y k k

R% k q l

R% ) 8A5 x

l 7 l 8

f l 8A5 p w y k u y k t t

l p w R% l 9 :

t t l X JPN3 1e 1 3

R% t t l

+ R%

k R% l ,

k R% - - l k

R% 0 u 5 l k

.e . u , 0 p p y u t l k p

u R% y l

k p 6 l

)-e )- k )*e )* k 1e 1 */ - Ak +) + Ak +- - A

p *- , A k u z w u u y u t

l y y k k R% u k

p R% u y u t l 1e 1

z , * T JU XU v w k t x R%

v w z y u t 7 l

Unfolding force

Contour length

Overall loss of water drives the folding of G-quadruplex

or i-motif in nanocage with reduced water activities