In e na ionale Ausgabe:DOI:10.1002/anie.201607965
G-Quad uplexes Deu sche Ausgabe:DOI:10.1002/ange.201607965
Ru hena iono Non-s acked Guanines in DNAG-Quad uplex
S uc u es:Enhancemen o c-MYC Exp ession
J8ssica Rod &guez, JesfflsMosque a, Jos8R. Coucei o,M.Eugenio V#zquez,* and
Jos8L. Masca eÇas*
Abs ac : Guanine quad uplexes(GQs) a e compac ou -
s anded DNAs uc u es ha playakey ole in he con ol o
a a ie y o biological p ocesses,including gene ansc ip ion.
Bulky u henium complexes ea u ing abipy idine,a e py -
idine,and one exchangeable ligand ([Ru( e py)(bpy)X]n+)a e
able o me ala e exposed guanines p esen in he GQ o he c-
MYC p omo e egion ha a e no in ol ed in quad uplex
base pai ing.qRT-PCR and wes e n-blo expe imen s indica ed
ha he complexes p omo e a ema kable inc ease in he
exp ession o his oncogene.Wealso show ha exchangeable
hioe he ligands (X=RSR’,Me ) allow egula ion o he
me ala ing ac i i y o he complex wi h isible ligh .
The e is g ea in e es in he de elopmen o me al-based
DNAbinde s ha show imp o ed selec i i y and educed
oxici y ela i e o cis-pla inum.[1] Ru henium complexes a e
p omising al e na i es owing o hei kine ic s abili y and ich
pho ochemis y and edox p ope ies.[2] In addi ion o non-
co alen ecogni ion o double-s anded DNA(dsDNA),
se e al u henium complexes ha o m co alen DNA
adduc s,especially h ough eac ion wi h he N7o guanines,
ha e also been desc ibed.[3] While mos o hese complexes
bind dsDNA, he unc ional ele ance o G-quad uplexes
(GQs)[4,5] calls o he de elopmen o p obes capable o
a ge ing hese s uc u es.[6] To ou knowledge, he e is only
one p eceden o he co alen me ala ion o aGQs uc u e
wi h a u henium complex, and he eac ion p esen s low
selec i i y.[7]
He ein, we demons a e ha coo dina ion complexes o
he ype [Ru( e py)(bpy)X]n+(X=Cl, RSR’,Me ) can
selec i ely me ala e unpai ed guanines p esen in pa allel
GQs,a eac ion ha is enhanced upon i adia ion. Impo -
an ly,weha e ound ha his selec i e me ala ion inc eases
he exp ession o he oncogene c-MYC,appa en ly by
dis up ing he pa allel GQ s uc u e p esen in i s p omo e
egion.
Ou wo k was concei ed a e lea ning ha while mos
DNA-me ala ing u henium agen s a e cy o oxic, he com-
plex [Ru( e py)(bpy)Cl]+(1)exhibi s e y low oxici y.[8] We
easoned ha he bulky and ela i ely hyd ophobic na u e o
his so o complexes could o e excellen oppo uni ies o
he selec i e modi ica ion o accessible guanines, he eby
p omo ing speci ic biological esponses wi h educed oxici y.
We i s s udied he abili y o chlo o complex 1 o
me ala e guanosine monophospha e (GMP). Mixing complex
1wi h 3equi o GMP in phospha e bu e (pH 7.5), led o
he pa ial o ma ion o he aquo complex [Ru( e py)-
(bpy)H2O]+2(2;o e 50%a e 30 min a RT), while he
guanosine emained essen ially un eac ed (Figu e 1B,
ace b). Fu he incuba ion o 2ha o ded he me ala ed
p oduc 3and he aquo de i a i e 2(Figu e 1A), wi h o al
consump ion o he s a ing chlo ide (Figu e 1B, ace d).
Figu e 1. A) GMP me ala ion eac ion wi h [Ru( e py)(bpy)Cl]PF6([Ru]-
Cl, 1)and o ma ion o he aquo de i a i e 2.B)HPLC o he eac ion
o 1(250 mm)and GMP (750 mm)in10mmphospha e bu e pH 7.5,
100 mmNaCl:in he da k a =0( ace a);a e 30 min in he da k
( ace b);ini ial mix u e a e 30 min o i adia ion a 455 nm ( ace c);
and ini ial mix u e a e 2hin he da k ( ace d). The injec ion peak is
labeled wi h an as e isk. C) Signal in he MS spec um co esponding
o he monoadduc 3.
[*] J. Rod &guez,D . J. Mosque a,D . J. R. Coucei o, D .M.E.V#zquez,
P o . J. L. Masca eÇas
Cen o Singula de In es igaciknenQu&mica Biolkxica eMa e iais
Molecula es (CIQUS), and Depa amen o de Qu&mica O g#nica
Uni e sidade de San iago de Compos ela
15782 San iago de Compos ela (Spain)
E-mail:j[email p o ec ed]
[email p o ec ed]
Suppo ingin o ma ion and he ORCID iden i ica ion numbe (s) o
he au ho (s) o his a icle can be ound unde h p://dx.doi.o g/10.
1002/anie.201607965.
T2016 The Au ho s. Published by Wiley-VCH Ve lag GmbH &Co.
KGaA. This is an open access a icle unde he e ms o he C ea i e
Commons A ibu ion-NonComme cial-NoDe i s License, which
pe mi suse and dis ibu ion in any medium, p o ided he o iginal
wo k is p ope ly ci ed, he use is non-comme cial and no modi ica-
ions o adap a ions a e made.
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In e es ingly,i adia ion o he ini ial mix u e o 30 min (l=
455 nm) led exclusi ely o he o ma ion o he monoadduc 3
(app oxima e con e sion o 80%based on he disappea ance
o GMP;Figu e 1B, ace cand Figu e S5 in he Suppo ing
In o ma ion). Impo an ly, he eac ion is ully o hogonal, so
compe i i e con ol expe imen s in he p esence o excess
adenosine monophospha e (AMP), cy idine monophospha e
(CMP), and hymidine monophospha e (TMP;Figu e S6), as
well as lysine o e en cys eine (Figu e S7), led exclusi ely o
he o ma ion o he GMP de i a i e.Mo eo e ,expe imen s
wi h double-s anded oligonucleo ides (dsDNAs) p esen ing
di e en a angemen s o guanines e ealed modes eac i -
i y,bu only wi h hose ea u ing e minal guanines (Table S1,
and Figu es S10–S13 in he Suppo ing In o ma ion). This
selec i i y mos likely s ems om he bulkiness o complex 1,
which canno each he s e ically hinde ed nucleophilic N7
si e in pai ed, in e nal guanines.[9]
Rema kably,i adia ion o amix u e o he pa allel c-
MYC quad uplex d[TTGAG3TG3TAG3TG3TA3][10] (10 mm)
wi h 5equi o 1in 10 mmphospha e bu e (pH 7.5) and
100 mmKCl led o he clean o ma ion o ap oduc wi h
amass co esponding o amonoadduc de i a i e (Fig-
u e 2A, ace C, peak a 22 min, MYC-[Ru],81% con e -
sion). We also obse ed eac ion in he absence o ligh
(app ox. 41%con e sion a e 30 min a RT,Figu e 2A,
ace b). Impo an ly,MSanalysis o he bo ine spleen
phosphodies e ase (BSP) diges ion o he u hena ed oligo-
nucleo ides lead o iden i ica ion o he i s guanine o he
sequence (5’-TTGA…, Figu e 2C)as he me ala ion si e
(Figu e S24).[11] No eac ion was obse ed unde he same
condi ions wi h amu a ed c-MYC GQ con aining aCins ead
o aGin posi ion 3(MYCm,d[TTCAG3TG3TAG3TG3TA3];
Figu e S18).
Thechemoselec i i y o he eac ion can be a ionalized
by conside ing he seconda y s uc u e o he c-MYC GQ
(Figu e 3), which shows ha G3 is exposed o he sol en and
does no pa icipa e in he o ma ion o he G-qua e s.As
aconsequence,i has ahighly accessible N7nucleophile ha
can eac wi h he bulky u henium complex. Impo an ly,
ci cula dich oism expe imen s e ealed ha he GQ secon-
da y s uc u e is dis up ed in he u henium adduc MYC-
[Ru] (Figu e S3, igh ). Con ol expe imen s wi h o he
ela ed pa allel quad uplex such as c-KIT1,which also
p esen s non-s acked guanines, e ealed asimila eac i i y
pa e n. Mass spec ome y shows ha he p omo e is
modi ied by he u henium complex a he expec ed posi ions
(Figu es S21 and S22), al hough cu iously,in his case,CD
analysis e ealed ha he quad uplex emained mos ly in ac
(Figu e S4).[12]
Wi h he abo e molecula in o ma ion, we explo ed
whe he Ru complex 1could a ec he exp ession o he
oncogene c-MYC,since his oncogene is in ol ed in many
impo an cellula p ocesses.[13] This was analyzed by eal-
ime quan i a i e e e se ansc ip ion PCR (qRT-PCR) in
HeLa and Ve o cells (Figu e 4and Figu e S26, espec i ely),
using po phy in TMPyP4, aGQbinde ha is known o
ep ess he exp ession o c-MYC,[14] as acon ol. As shown in
Figu e 4, HeLa cells ea ed wi h 1(100 mm)inDulbeccoQs
modi ied EagleQsmedium (DMEM) p esen ed amodes bu
signi ican inc ease in he ansc ip ion o c-MYC compa ed
o un ea ed cells (80%a 16h and 200%a 48h).[15] As
expec ed, ea men wi h TMPyP4 led o a60% dec ease in
he cellula le els o c-MYC mRNAa e 48 h.
We also analyzed he exp ession o c-MYC p o ein by
wes e n blo . In ag eemen wi h he qRT-PCR esul s, ea -
men o cells wi h 100 mmo 1,led o ano iceable inc ease in
he le els o c-MYC p o ein (Figu e 4, a e aged 40%
Figu e 3. S uc u e o he GQ o c-MYC (PDB ID:1XAV), highligh ing
he exposed G3and he me ala ion selec i i y.The la ge sphe es in he
quad uplex s uc u e ep esen po assium ions.
Figu e 2. A) HPLC aces o amix u e o he GQ o c-MYC (MYC,
10 mm)and 1(5 equi ) in 10 mmphospha e bu e pH 7.5, 100 mm
KCl, a RT:in he da k a =0( ace a);a e 30 min in he da k
( ace b), ini ial mix u e a e i adia ion o 30 min a 455 nm ( ace c).
B) MS o he me ala ed p oduc (MYC-[Ru]) showing he peaks
co esponding o he complex (m/z=8089) and he deme ala ed
agmen (m/z=7600). C) MS o he p oduc s a e BSP diges ion o
MYC-[Ru] showing he peaks co esponding o he diges ed complex
(m/z=7489) and non-me ala ed diges ed p oduc (m/z=7000) esul -
ing om he clea age a G3as shown by he dashed line in he
s uc u e.
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inc ease). These da a con i m ha , in con as o mos
quad uplex a ge ing agen s,complex 1p omo es an inc ease
ins ead o adec ease in he le el o gene exp ession, he eby
ac ing as a ansc ip ional ac i a o .[16] ICP-MS measu emen s
o isola ed nuclei and ch oma in ob ained a e ea men
wi h complex 1con i med he p esence o ela i ely signi i-
can amoun s o u henium, which is consis en wi h e icien
cellula up ake and nuclea deli e y o he complex
(Tables S2 and S3). In ag eemen wi h he ea ly s udies,[8]
cell iabili y assays con i med ha 1is essen ially non-
cy o oxic (Figu e S27).
While he abo e da a indica e ha 1is capable o al e ing
he exp ession o c-MYC, he de elopmen o de i a i es ha
could be ac i a ed by using ex e nal i adia ion would be
highly a ac i e.[17] Towa ds his aim, we p epa ed complexes
4and 5,which ea u e a hioe he ligand (Figu e 5) and a e
kine ically s able bu unde go apid ligand exchange upon
i adia ion wi h isible ligh .[18] In con as o he chlo ide
complex 1,in i o expe imen s wi h he hioe he de i a i e
4showed ha i does no eac wi h he c-MYC quad uplex in
he da k a e 30 min, bu yields he desi ed GQ monoadduc
upon i adia ion (71%con e sion, Figu e S17). Theace yl-
me hionine de i a i e 5p esen s e en highe kine ic s abili y
han complex 4,and no aces o he me ala ion adduc s,o
e en o he aquo de i a i e 2,we e obse ed a e se e al
hou s in he da k. Howe e ,i adia ion o 30 min igge s
e icien co alen me ala ion o he GQ (Figu e S20). We also
analyzed he e ec o complex 5on ansc ip ion o he c-
MYC gene by qRT-PCR in Hela cells.Asshown in he
Figu e 5(le ), c-MYC mRNAle els inc eased a e i adi-
a ion, becoming simila o hose obse ed a e ea men
wi h he aquo complex 2.Wes e n-blo analysis con i med
ha enhancemen o he gene exp ession only ook place in
he p esence o ligh , while he le els o he p o ein c-MYC
did no change when he cells we e kep in he da k (Figu e 5
and Figu e S29). As expec ed, bo h qRT-PCR and wes e n-
blo expe imen s showed ha he aquo compound 2is ac i e
bo h in he da k and unde i adia ion. Mo eo e ,con ol
expe imen s con i med ha he i adia ion does no ha e any
measu able e ec on cell iabili y (Figu e S28), and ha he
complex does no gene a e signi ican amoun s o single
oxygen upon i adia ion (Figu e S25).
In conclusion, we ha e demons a ed he selec i e modi-
ica ion o accessible guanines lanking he GQ o c-MYC
wi h designed bulky u henium complexes.Impo an ly, he
bioo hogonal me ala ion enhances he exp ession o he
oncogene c-MYC.Gi en ha an inc ease in c-MYC an-
sc ip ion has been shown o be impo an in se e al cance s,in
pa icula o he enewal abili y o cance s em cells,[19] ou
disco e y migh lead o in e es ing biological applica ions.
Acknowledgemen s
We hank he suppo by he Spanish g an s SAF2013-41943-
R, CTQ2015-70698-R, and CTQ2013-49317-EXP, he Xun a
de Galicia GRC2013-041, he ERDF,and he Eu opean
Resea ch Council (Ad anced G an No.340055). Suppo o
COST Ac ion CM1105, COST CM1306 and he o eo-cinqa
ne wo k a e also kindly acknowledged. J.R. hanks he Xun a
de Galicia o aPh.D. ellowship.We hank Rebeca Menaya
Figu e 4. A) qRT-PCR analysis o c-MYC (ligh ba s) and ALAS1 (black
ba s) ansc ip ion. Hela cells we e incuba ed o 16 ho 48hwi h
ei he 100 mmRuCl, TMPyP4, o 50 mm5- luo ou acil(5-FU, adis up o
o RNA syn hesis). Amoun s a e ela i e o he exp ession le els o he
housekeeping gene GAPDH.Values a e he a e age o h ee expe i-
men s, and e o ba s indica e he s anda d e o .B)Analysis o he
exp ession o c-MYC by wes e n-blo . HeLa cells we e incuba ed o
16 hwi h 100 mmcompound 1and hen lysed, and c-MYC was
de ec ed by SDS-PAGE, ollowed by wes e n blo wi h an an i-MYC
An ibody (uppe panel). The ela i e amoun o p o ein in wo
independen expe imen swas quan i ied by densi ome y (lowe
panel). Da a a e ep esen ed as he old change wi h espec o
un ea edcon ols. E o ba s indica e he s anda d de ia iono he
old change wi h espec o un ea ed con ols.
Figu e 5. A) Thioe he complexes 4and 5.B)c-MYC ansc ip ion as
measu ed by qRT-PCR in he da k (black ba s) o a e i adia ion
(60 min, ligh ba s). Hela cells we e incuba ed o 16 hwi h 100 mm
RuMe (5)o aquo complex 2.RNA le els a e gi en ela i e o he
exp ession le els o he housekeeping gene GAPDH.C)Exp ession o
c-MYC as measu ed by wes e n blo in he p esence o absence o
compound 5.The ela i e amoun o p o ein wi h espec o ha o b-
ac in was quan i ied by densi ome y (lowe panel). The expe imen al
p ocedu es a e as desc ibedinFigu e 4.
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o he help wi h he biological expe imen s and Ve lnica
PiÇei o o he Unidad de An#lisis Elemen al (RIAIDT-USC)
o he ICP analysis.
Keywo ds: bioino ganic chemis y ·DNA damage ·me ala ion ·
pho ochemis y · u henium
How oci e: Angew.Chem. In . Ed. 2016,55,15615–15618
Angew.Chem. 2016,128,15844–15847
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Manusc ip ecei ed:Augus 17, 2016
Re ised: Sep embe 29, 2016
Final A icle published: No embe 17, 2016
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15847Angew.Chem. 2016,128,15844 –15847 T2016Die Au o en. Ve ç en lich on Wiley-VCH Ve lag GmbH &Co. KGaA, Weinheim www.angewand e.de