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東北大学大学院理学研究科化学専攻
分析化学研究室

１９１８年（大正７年）創立： 最も伝統ある分析化学研究室

分析化学とは？
分離・精製・検出法の開発
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♦ NMR♦ 質量分析法・・・田中博士♦ PCR法♦ クラウンエーテル♦ 緑色蛍光タンパク・・下村博士♦ ......

「化学入門コース」より、
梅澤喜夫・大野公一・竹内敬人 編、岩波書店

新規な分析装置 or 分子プローブを研究・開発する学問！

COMMUNICATION

Cite this: Chem. Commun., 2019,55, 3183

Trimethine cyanine dyes as deep-red fluorescentindicators with high selectivity to the internalloop of the bacterial A-site RNA†

Received 17th January 2019,
I\>N\FM
Yusuke Sato,
Accepted 29th January 2019

Full Paper]J)%7-:&Sayaka Yajima, Akifumi Taguchi, Kyosuke Baba,
Mayu Nakagomi,
FULL
PAPER
Yuri
AibaandSeiichi
Nishizawa*蛍光インジケーター�
両親媒性α-ヘリックスペプチド�

.!"[Q<@DOI: 10.1039/c9cc00414a& Probes

Tight binding to bacterial A-site: 2Amino-5,6,7-trimethyl-1,8-naphthyridine(ATMND) possessing an aminoethyl side
chain was developed as the strong andselective binder to the internal loop of
the bacterial A-site. This ligand shows
the tightest binding reported to dateamong non-aminoglycoside ligands.
Also, this ligand was demonstrated tobe applicable to the fluorescence indicator displacement assay for assessing
ligand/A-site interactions.

'4#8190SP;^

Y. Sato, M. Rokugawa, S. Ito, S. Yajima,
H. Sugawara, N. Teramae, S. Nishizawa*

rsc.li/chemcomm&& – &&
Fluorescent Trimethylated
Naphthyridine Derivative with an
Aminoalkyl Side Chain as the Tightest
Non-aminoglycoside Ligand for theBacterial A-site RNA21+,?K19021+,190
We report that TO-PRO-3, a thiazole orange analogue witha trimethinebridge, functions as a deep-red fluorescent indicator for theN[L7$:,?Kinternalloop structure of the bacterial (Escherichia coli) ribosomal decoding

1*3

エクソソーム�region of the aminoacyl-tRNA site (A-site), which enables the assessment of A-site binding capability of various test compounds including
blue and even-green-emitting compounds.

I\W!5(:&HAVHRTUFM

With increasing knowledgeRNA選択的蛍光色素�
about the diverse roles of RNAs within
三重鎖形成ペプチド核酸プローブ�
the cells, targeting RNA structures with small ligands has received
1N
considerable attention in drug discovery. Aminoacyl-tRNA site
SONH
O
NH
ONH
NH
NH(A-site) of the 16S RNA decoding region in the bacterial ribosome
NHNNH
NNH
N
N
NN
O
N
O
N
O
NON
N
O
NO
N
O
N
O
represents one of the most validated RNA targets.1,2 Aminoglycosides
OO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
are well-characterized ligands that can strongly and selectivelybind
H N
NNN
N
N
N
N
NNNNNNNN
N
NNNH
HHHH
HHHH
Hto the asymmetric internal loop formed by three adenines in thebacterial A-site (Fig. 1A).3 This causes mistranslation or prematureNHYI\>EBCX=Q/ 89:KFM
termination of protein synthesis,4 which ultimately results in the5OI\[<63:GZD[Qbacterial cell death. Althoughaminoglycosides are widely used as
ペプチドプローブ・膜タンパク結合アプタマー�
antibiotics in the treatment of the bacterial infections, they possess
several undesirable properties for clinical use, such as the emergence Fig. 1 (A) Sequence of the bacterial (Escherichia coli) A-site-containingof resistant bacterial strains and oﬀ-target eﬀects.6 In order RNA model used in this study. The sequence inside the box corresponds tothe decoding region of E. coli 16S A-site. The sequence numbers of the
私たちの研究室では、RNAや細胞外小胞の機能を解明するための分子プローブを創製し、
to circumventthese concerns with aminoglycosides, it is ofE. coli 16S RNA are indicated in parentheses. Chemical structures ofgreat importanceto develop new A-site-binding ligands whose TO-PRO-3, TO and TO-PRO-1 are also shown. (B) Schematic illustration
これらに基づく検出・疾患診断・機能解析法や創薬の支援ツールを提案・開発することで、structures are distinct from the aminoglycosides.7,8of a FID assay using TO-PRO-3 as a deep-red indicator for A-site RNA.
生命科学の発展に貢献することを目指しています！
In this context, the fluorescenceindicator displacement(FID) assay has been useful for the screening of RNA-binding
Aminoglycosides tagged with a fluorophore (pyrene orligands because neither covalent labeling nor immobilization+22222222

to exhibit
higherresponsivenessfor fordsRNAsoveroverthe then ain96-wellplateplatefor 18likelyto exhibit
higherresponsivenessdsRNAseda 96-wellfor h18before
h before likely
dsDNAs,whichwouldpartly
account
for forthe them
was wasthenthenexchanged
by diﬀerent
corresponding
dsDNAs,whichwouldpartly
account
diumexchanged
by diﬀerent corresponding
RNARNA
selectivity(cf.,(cf.,Figure1B).1B).
NoteNotethatthat
BIQBIQainingmediumsolutions.
Theyobserved
selectivity
Figureontaining
mediumsolutions.
They observed
the thepronounced
light-up
response
for bothrG and
dG dG
5%
CO2CO
for2 for20 minbeforecell cell showed
showedpronounced
light-up
response
for bothrG andin 5%
20 minbeforecould
adopt
G-quadruplex
structures(FigureS9).S9).
ThisThishealamarBlue
assayassay(Invitrogen,couldadopt
G-quadruplex
structures(Figurey thealamarBlue(Invitrogen, thatthat
resultfromfrom
BIQBIQbinding
to tothese
G-quadruplex
um solutions
werewereexchanged
by by wouldwouldresult
bindingthese
G-quadruplex
mediumsolutions
exchanged
生細胞RNAイメージングのための蛍光色素
as observed
for forits its
analogous
thiazoleorangeowed
by theadditionof 10
structures,
as observed
analogous
thiazoleorangefollowedby theaddition
of μL
10 of
μL of structures,
35 35(TO).
well.
AfterAfterincubationfor 3forh,3theBIQBIQ
showedalmostno noﬂuorescence(TO).In addition,
ch well.
incubationh, the シアニン色素の構造を最適化することで、細胞中のRNA（核小体）を選In addition,
showedalmostﬂuorescence
cell cell
plateplate
at 580nm nm(excitationfor proteins
and andsmallsmall
biomolecule(FigureS10).
each
at 580(excitation response
response
for proteins
biomolecule(FigureS10).
択的に染色できる蛍光色素BIQを開発しました。BIQは、「生きた細胞に
a microplate
reader(SpectraMaxWe We
examined
the theRNARNA
stainingabilityof BIQ(5.0(5.0μM)μM)
for forwitha microplate
reader(SpectraMax
examined
staining
abilityof BIQ
適用でき」、かつ「明瞭な発蛍光応答を示す」世界トップレベルの深赤色蛍
living
MCF7cells,cells,
along
withwiththe thecostaining
withwith
nuclearCA).
livingMCF7
along
costaining
nuclear
光RNAイメージング色素です。
selective
Hoechst33342,
a blue-emissiveDNAstaining
dye.dye.
selective
Hoechst33342,
a blue-emissiveDNAstainingSSION
SCUSSION
AfterAfterincubation
withwith
BIQBIQfor for20 min,the theemission
in theincubation
20 min,emissionin the
近年、核小体は、ウイルス感染やオートファジー、細胞老化との関連が
deep-redregionappearedin thecytoplasm
and andnucleolus
deep-redregionappearedin thecytoplasm
nucleolus
nce responseof BIQ(1.0(1.0μM)μM)
to to注目されており、BIQは核小体の機能研究に役立つことが期待できます。
escence
responseof BIQ(Figure2A).2A).
ThisThis
indicates
goodgood
cell cell
plasmaand andnuclear(Figureindicatesplasmanuclearymus
DNAor E.orcoli
totaltotalRNA,thymusDNA
E. coliRNA,さらに、現在、BIQを応用することで、ウイルスRNA検出法や抗ウイルス
sodiumphosphatebuﬀer(pH(pH薬のスクリーニング法の開発を進めています。
0MmM
sodiumphosphatebuﬀer
BIQ
missionin theabsenceof nucleic
noemission
in theabsenceof nucleic
umablydue dueto thenonradiative
resumablyto thenonradiativearoundmonomethine
bridgeon aroundthemonomethine
bridge(b) BIQ
(c) Hoechst33342 （図の解説）(a) theMergedBIQによる生細胞（ヒト乳がん細胞）のquinoline
rings,
like likemany
end andquinoline
rings,
many
蛍光イメージング：核（青色）の中で、赤sorptionand andemission
spectraof of
Absorption
emission
spectrabetween
5.8 and8.0 (FigureS5).S5).
pH
between
5.8 and
8.0 (Figure
く染まっている部分が核小体
s the thesigniﬁcant
increasein theauses
signiﬁcant
increasein theQBIQ
in thedeep-red
spectralregionin thedeep-red
spectralregion
この研究成果は、
ght-upfactor(I/I(I/II andI0 I0e light-up
factorI and0, where0, where
H C
アメリカ化学会誌
Nsities in theand andabsencentensities
in presence
the presence
absenceN
Hoechst33342
（Anal. Chem.）のN
eached105-fold.
Weobserved) reached 105-fold. We observed
Supplementary
NN
H
iftof
theabsorption
bandofBIQ
d shift of the absorption band of BIQ
Cover Art にハイN
HOCHCH
ライトされましegureS6),S6),
suggesting
theintercalative
suggesting the intercalative
た！
the theobserved
light-up
responseus,observed
light-up
responseN
dictedrotationof BIQby intercalation
rotationof BIQby intercalation
15 µm
Anal. Chem., 91,
BIQ
A.
Signiﬁcantly,
thelight-up
factorRNA. Signiﬁcantly, the light-up
factorN
14254 (2019)oflargerthanthanto thoseersmagnitudeof magnitudelargerto those
14(5-fold)14 thatwere previously

DNA staining dye.
he emission in thesm and nucleolus
asma and nuclear

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東北大学大学院理学研究科化学専攻
分析化学研究室
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