Double‐Stranded RNA‐Specific Templated Reaction with Triplex Forming PNA

TLDR: Double‐stranded RNA‐specific templated reaction resulting from PNA‐reagent conjugates that are brought within reactive distance through the formation of sequence‐specific triplexes onto double‐Stranded RNA is reported.

Abstract: RNA, originally perceived as a simple information transfer biopolymer, is emerging as an important regulator in cellular processes. A number of non‐coding RNAs are double‐stranded and there is a need for technologies to reliably detect and image such RNAs for biological and biomedical research. Herein we report double‐stranded RNA‐specific templated reaction resulting from PNA‐reagent conjugates that are brought within reactive distance through the formation of sequence‐specific triplexes onto double‐stranded RNA. The reaction makes use of a ruthenium‐based photocatalyst that reduces a pyridinium‐based immolative linker, unmasking a profluorophore. The reaction was shown to proceed with signal amplification and to be selective for double‐stranded RNA over DNA as well as single‐stranded RNA. The generality of the triplex formation was enabled by non‐canonical nucleobases that extend the Hoogsteen base‐pairing repertoire. The technology was applied to a templated reaction using pre‐microRNA 31.

Figures

Figure 5. The structure of pre-miR-31hairpin (71-mer) and possible binding site for PNAs. Pre-miR-31 was divided into two parts, miR-31-5p and -3p sequences for this work.

Figure 6. One-to-one templated reaction of (A) 5pRu1+5pCou1 (100 nM), (B) 5pRu1+5pCou2 (100 nM), and (C) 5pRu2+5pCou3 (100 nM) in the presence of the duplex or single strand form of mir-31-5p and 3p (100 nM); (D) Templated reaction between 5pRu2 (50 nM) and 5pCou3 (250 nM) in the presence of 12.5-100 nM of pre-miR-31 duplexes formed by miR-31-5p and 3p. Conditions: 1´PBS buffer, 0.02 % tween-20, 5 mM sodium ascorbate, 140 min. of reaction time, 1% of sperm DNA.

Figure 1. Top: Schematic representation of dsRNA templated reaction between Ru(bpy)2phen or pyridinium coumarin (PyCou) based on triplex formation leading the accumulation of fluorophore (Fl); Bottom: Chemical structures and Hoogsteen hydrogen-bonding patterns of modified nucleobases M, P, and E designed to recognize the G-C, C-G, and U-A nucleobases, respectively, as well as T which pairs A-U for a PNA-dsRNA triplex.

Figure 2. Top: RNA sequences used and alignment of the PNA probes (see SI for explicit structures); (A) Ru1+Cou1, (B) Ru1+Cou2, and (C) Ru1+Cou3 in the presence of RNA1+RNA2, RNA1, RNA2, or none. peg = [(aminoethoxy)ethoxy] acetic acid, K = lysine. Reaction condition: 100 nM of PNAs and 100 nM of ss or dsRNAs, 30 mM HEPES-KOH pH 6.85, 50 mM NaCl, 5 mM sodium ascorbate, 0.02 % Tween-20; (D) Measured half-life and kapp of the templated reactions.

Figure 4. (A) Templated reaction of Ru2+Cou4 in the presence of RNA1+RNA2 with different concentration of NaCl (background signal is from ssRNA or none); (B) templated reaction of Ru2+Cou4 in the presence of fully matched dsRNA (RNA1+RNA2) or mismatched dsRNA sequence (RNA3+RNA4) in 100 mM NaCl concentration. Reaction condition: 100nM of PNAs and 100 nM of ss or dsRNAs, 30 mM pH 6.85 HEPES-KOH, 5 mM sodium ascorbate, 0.02 % Tween-20, incubation time: 30 min.

Figure 3. (A) Plot of the conversion for the templated reaction of Cou3 (250 nM) with 1 eq. (250 nM, black line) or 0.2 eq. of Ru1 (50 nM, red line) in the presence of 0.2 eq. (50 nM) of dsRNA (RNA1+RNA2) or ssRNA; (B) Plot of the conversion for the reaction at various template (dsRNA: RNA1+RNA2) loading; (C) Calculated yield and turnovers for the reactions (the yield was obtained by subtracting the conversion observed in the presence and absence of template). Reaction condition: 30 mM HEPES-KOH pH 6.85, 50 mM NaCl, 5 mM sodium ascorbate, 0.02 % Tween-20. Yields are calculated based on a titration of the coumarin, see SI for details.

Full text

Article
Reference
Double-Stranded RNA-Specific Templated Reaction with Triplex
Forming PNA
KIM, Kitae, CHANG, Dalu, WINSSINGER, Nicolas
Abstract
RNA, originally perceived as a simple information transfer biopolymer, is emerging as an
important regulator in cellular processes. A number of non‐coding RNAs are double‐stranded
and there is a need for technologies to reliably detect and image such RNAs for biological and
biomedical research. Herein we report double‐stranded RNA‐specific templated reaction
resulting from PNA‐reagent conjugates that are brought within reactive distance through the
formation of sequence‐specific triplexes onto double‐stranded RNA. The reaction makes use
of a ruthenium‐based photocatalyst that reduces a pyridinium‐based immolative linker,
unmasking a profluorophore. The reaction was shown to proceed with signal amplification and
to be selective for double‐stranded RNA over DNA as well as single‐stranded RNA. The
generality of the triplex formation was enabled by non‐canonical nucleobases that extend the
Hoogsteen base‐pairing repertoire. The technology was applied to a templated reaction using
pre‐microRNA 31.
KIM, Kitae, CHANG, Dalu, WINSSINGER, Nicolas. Double-Stranded RNA-Specific Templated
Reaction with Triplex Forming PNA. Helvetica Chimica Acta, 2018, vol. 101, no. 3, p.
e1700295
DOI : 10.1002/hlca.201700295
Available at:
http://archive-ouverte.unige.ch/unige:103235
Disclaimer: layout of this document may differ from the published version.
1 / 1

1
!"#$%&'()*+,-&-./01'23&45654.7&83%+)&-./&+4)5",.95):.7*53%&;.<"*85,=.>01.
Ki#Tae#Kim,#Dalu#Chang,#and#Nicolas#Winssinger#*
,
#
Department#of#Organic#Chemistry,#NCCR#Chemical#Biology,#Faculty#of#Science,#University#of#Geneva,#30#quai#
Ernest#Ansermet,#1211#Gene va ,#S witzerland .##
#
1?(7/1@7A#RNA,#originally#perceived#as#a# simple#information#transfer#biopolymer,# is# em erging# as#an#important#
regulator# in# cellular# processes.# # A# nu m b er# of# non-coding# RNAs# are# d ouble-strande d# and# there# is# a# need# for#
technologies#to#reliably#d etect#and#image#such#RNAs#fo r#biological#and#biomedical#research.#Herein#w e#report#
double-stranded# RNA-specific# templated# reaction# resulting# from# PNA-rea gen t# con jug ates # that# are# brought#
within#reactive# distance# through#the#formation# of#sequence-specific#triplexes#onto#double -strand ed#RNA.##The#
reaction# make s# us e# of# a # ruth en ium -based# photocatalyst# that# reduces# a# pyridinium-based# imm olative# linker,#
unmasking#a#profluorophore.##The#reaction#was# shown# to# proceed#with# signal#amplification#and# to# be#selective#
for# do u ble-stranded# RNA #over# DNA#as#w ell# as# single-stranded#RNA.##Th e#generality# o f#the# triplex#formation#was#
enabled#by#non-canonical#nucleobases#that#extend#the# Ho ogsteen# b ase-pairing#repertoire.##The#technology#was#
applied#to#a#templated#reaction#using#pre-microRNA#31.#
B&C9"*-2:#Template d #re a ct io n #• #su p r amolecular#c h e mistry#•#PNA #• #d sR NA#•#miR#
#
D,)*"-# 4 )5" ,.
The#past#decades#have#brought#a#paradigm#shift#regarding#the#role#of#RNA#with#accumulating#evidence#that#its#functions#extend#far#beyond#
simple#messaging#between#DNA#and#proteins.
[1]
##Only#3#%#of#our#genome#encodes#proteins,#yet#the#work#that#emerged#from##the#
Encyclopedia#of#DNA#Elements#(ENCODE)#project
[2]
#revealed#that#76%#of#the#genom e#is#tran scrib ed ,#bolsterin g#the #notio n#th at#an #im po rtan t#
portion#of#non-coding#RNA#(ncRNA)#has#a#role.##Non-coding#RNA#(ncRNA)#genes#yield#functional#RNAs#rath er#than #pro teins,#w ith#im po rtan t#
function#in#directing#post-translatio nal#regu lation #of#gen e#exp ressio n#an d#R NA #m od ifications .#
[3]
#Double-stranded#RNA#(dsRNA)#has#emerged#
as#an#important#regulator#of#gene#expression#in#many#eukaryotes.#It#triggers#different#types#of#gene#silencing#that#are#collectively#referred#
to#as#RNA#silencing#or#RNA#interference#.
[4]
#Such#ncRNAs#are#typically#regulated#by#a#complex#set#of#modifications#and#understanding#their#
biogenesis#is#crucial.
[5,#6]
#As#a#single#strand#biopolymer,#RNA#tends#to#adopt#diverse#intermolecular#folds#with#stretches#of#dsRNA.
[7]
##
MicroRNAs#(miRs)#which,#in#their#mature#form#are#approximately#21#nt,#are#processed#from#larger#double-stranded#precursors#following#a#
choreographed#series#of#events.#
[8,#9]
#These#advances#have#transformed#our#appreciation#for#the#tremendous#number,#diversity#and#
biological#importance#of#ncRNAs.##Accordingly,#technologie s#to#se n se#a nd #in terfe re#w ith #ncR N A s#are #im po rta nt.
[10]
#Recently,#triplex-forming#
PNAs#with#a#fluorogen#as#a#base#surrogate#were#reported#as#a#turn-on#probe#for#dsRNA.#
[11,#12]
##Oligonucleotide-templated#reactions#have#
emerged#as#a#powerful#technology#to#sense#and#image#ssDNA#or#RNA.
[13-17]
#Oligonucleotide-templated#reactions#are#prom o ted#b y#the#h igh#
effective#concentration#achieved#following#hybridization#of#the#reagent#conjugates.##These#reactions#have#been#shown#to#be#possible#in#a#
cellular#context#as#well#as#live#organisms
[18]
#and#can#be#used#to#unmask#fluorophores#or#bioactive#compounds.
[19-21]
#Templated#reactions#
have#the#potential#to#turnover#and#provide#signal#amplification.##Proteins#have#also#been#used#to#template#reactions#using#the#same#
concept#of#proximity-induced#reactio ns .
[22-24]
###Herein#we#extend#this#che m ist ry#to #d sR N A#a n d#d e m o n stra te #th at#t he #rea ct ion #is#specific#to#a#
dsRNA#over#dsDNA#or#ssRNA#(Fig.#1).#
While#peptide#nucleic#acids#(PNAs)#are#known#to#form#triplex#with#DNA,
[25]
#such#triplex#formations#are#restricted#to#purine#sequences#
using#the#canonical#nucleobases#and#proceed#under#specific#conditions#(low#salt,#acidic#pH).##Recently,#Rozner#and#coworkers#extended#the#
scope#of#triplex#forming#PNA#with#the#introduction#of#monomers#bearing#novel#nucleob ases#that#extend #the#Hoogstee n#triplex#base-
pairing
[26]
#to#any#sequence#permutation #(M •G-C,#P•C-G,#E•U-A,#Fig.#1)#and#showed#that#triplex#formation#was#selective#for#dsRNA#over#
dsDNA.
[27-30]
#The#fact#that#the#M#nucleobase#is#more#basic#than#the#C#nucleobase#also#enables#triplex#formation#at#higher#pH,#at#or#near#
physiological#conditions.###

2
#
<5=#*&.EF#Top:#Schematic#representation#of#dsRNA#templated#reaction#between#Ru(bpy)
2
phen#or#pyridinium#coumarin#(PyCou)#based#on#triplex#formation#
leading#the#accumulation#of#fluoroph o re#(Fl);#Bottom :#C h emical#structures#and#Hoogsteen#hydrogen-bonding#patterns#of#modified#nucleobases#M,#P,#and#E#
designed#to#recognize#the#G-C,#C-G,#and#U-A#nucleobases,#respectively,#as#well#as#T#which#pairs#A-U#for#a#PNA-dsRNA#triplex.#
/&2#%)2.+,-.!524#225",.
Design'of'dsRNA'templated'reaction'
We#began#our#work#with#a#dsRNA#derived#from#sequences#that#have#been#productively#used#in#hybridization#chain#reaction#(HCR).
[31]
#As#
shown#in#Fig.#1;#two#PNA#probes#conjugated#to#the#ruthenium-based#photocatalyst#and#the#pyridinium-based#immolative#linker,#
respectively,#are#required.##Triplex#formation #brin gs#the #ruthen ium-based#photocatalyst#(Ru(bpy)
2
phen)#within#reactive#distance#of#the#
pyridinium-based#immolative#linker.##Photoexcitation#of#the#catalyst#using#a#455#nm#LED#lamp#followed#by#ascorbate#reduction#yields#a#
reduced#ruthenium#catalyst#that#tra nsfe rs#an #electron #to#the #pyrid iniu m #resultin g#in#a n#elim in atio n#of#th e#ben zylic #sub stituent#(immolation).##
We#opted#for#difluorocoumarin#as#the#leaving#group#based#on#the#fact#that#it#had#been#successfully#used#in#the#templated#reaction#
previously,#yielding#a#fluorogenic#signal#that#is#spectrally#resolved#from#the#ruthenium#photocatalyst.
[32]
##The#design#of#the#probes#was#
made#with#the#following#considerations:#each#probe#should#use#a#different#strand#of#the#duplex#to#minimize#any#background#reaction#
arising#from#ssRNA-templated#reaction;##sequences#rich#in#M#and#T #m on om ers #will#form #m ore #stab le#triplex;#8-mer#probes#should#achieve#
the#necessary#affinity#to#yield#a#templated#reactio n#at#low #co nce ntra tion#(less#th an 10 0#nM );#the #reage nts#sh ou ld#be#se pa rated#from#the#
PNA#with#a#short#polyethylene#glycol#spacer#(PEG:#9#atoms)#to#relieve#any#unfavorable#conformational#bias.##Based#on#t h ese #co n sid era tio n s#
a#set#of#probes#was#designed#as#shown#in#Fig.#2.##The#ruthenium#photocatalyst-conjugate#probe#(/#E)#interacts#with#/01.E#of#the#
/01E:/01G#duplex,#whereas#the#coumarin-conjugate#probes#(@"#E-H)#interact#with#/01G#of#the#same#duplex.##Three#different#coumarin-
conjugate#probes#were#prepared#in#order#to#vary#the#distance#between#the#reaction#sites.#
The#reactions#were#monitored#through#the#increase#of#fluorescence#arising#from#the#unmasking#of#coumarin#as#a#function#of#time.##
Using#optimal#conditions#for#triplex#formation#(pH#6.85,#HEPES-KOH#buffer,#50#mM#NaCl)#and#performing#the#reaction#at#100#nM#of#probes#
with#stoichiometric#template,#we#were#pleased#to#observe#a#dsRNA-specific#reaction#(Fig.#2a).##The#reaction#of#/#E#and#@"#E#proceeded#
significantly#faster#in#the#presence#of#dsRNA#than#with#either#of#the#single#strand#RNA#(/01E#or#/01G).##Comparing#the#initial#speed#of#the#
reaction#from#the#slope#of#the#reac tion#a fter#10 #m in,#the#d sRN A#w a s#7#tim es #faster#th an#eith er # sin gle#strand#RNA#and #71#times#faster#than#
the#background#reaction#lacking#RNA #tem pla te.##It#is#notew orth y#tha t#the#high #selectivity#fo r#dsR NA #vs#single#strand#RNA#templated#reaction#
is#dependent#on#th e#s alt#c o nc en tra tio n ;#in#th e #ab se n ce#o f#N a Cl,#th e #rea ction#of#dsRNA #w as#o nly#2 - fo ld#faste r#tha n#ssRNA#(see#Fig.#S1,#A).##At#
concentration#of#NaCl#above#50#mM,#the#reaction#had#comparable#pe rforman ce#as#50#mM #bu t#with#slower#kinetics#(Fig.#S1#B#and#C#for#60#
and#70#mM#NaCl,#respectively).##Importantly,#the#reaction #also#a fford ed#go od #discrim ina tion#b etw een #dsR NA #an d#ssR NA #tem pla te#in#PB S#
buffer#(Fig.#S1#D).##
We#next#compared#the#reactivity#of#coumarin#probes#leaving#a#single#or#double#base-pair#gap#between#the#probes#forming#the#
triplex#(@"#G#and#@"#H#respectively).##Both#of#these#reactions#pro ved #to#be#a lm os t#twice #as#fas t#com p are d#to#the #reactio n#o f#@"#E#+#/#E#
(Fig.#2#B#and#C),#with#an#initial#rate#of#reaction#that#is#over#10-fold#faster#for#dsRNA#than#the#reaction#w ith#eithe r#ssRN As .##A#dsD N A#
template#with#the#same#sequen ce#as #/01E# +#/01G#did#not#catalyze#the#reaction,#nor#did#either#of#the#ssDNA#template#(Fig.#2C).###Assuming#
a#quantitative#formation#of#the#quaternary#complex#of#the#dsRNA#with#the#two#probes#forming#the#triplex,#the#reaction#is#anticipated#to#
follow#a#first#order#kinetics#and#the#half-life #o f#th e #rea ctio n #ca n#b e #us ed #to #d eriv e#a #ps eu d o #first-order#rate#constant.##Following#this#analysis,#
rates#of#0.44#x#10
-3#
to#0.89#x#10
-3
#s
-1
#were#measured#for#the#different#reactions#(Fig.#1D).##Comparing#the#sequence#of#@"#E#vs#@"#G,#they#

3
have#the#same#nucleobase#content,#suggesting#that#the#2-fold#kinetic#difference#observed#between#the#reactions#of#/#E#with#@"#E#vs#@"#G#
is#the#fact#that#there#is#a#m o re #fa vo rab le#reagent#alignment#in#the#latter#reaction.###Another#possible#explanation#is#the#fact#that#adjacent#M#
nucleobases#at#the#junction#of#@"#E#and#/#E#triplex#with#protonated#amino#pyridines#result#in#a#distortion#that#is#slightly#detrim en tal#to#the #
reaction#kinetics.##The#sam e#d ep e nd e n ce#o f#N a C l#co nc en tra tio n #w as#o b se rve d#f or#t he #re act ion #o f#@"#H#and#/#E#as#with#@"#E#and#/#E#with#
respect#to#the#selectivity#of#dsRNA#templa ted #reactio n#vs#ss RN A#templated#reaction.#Namely,#50#mM #Na Cl,#or#h ighe r#conc entra tions,#is#
important#to#achieve #go o d #se lect ivity#o f#d s RN A #vs#s sR N A#( Fig .#S2 #A,B ).###Importantly,#the#reaction#of #@"#H#with#/#E#proceeded#equally#at#pH#
7.4#with#a#high#selectivity#for#dsRNA##(Fig.#S2#C,D).##Performing#the#reaction#at#different#concentrations#(100#–#400#nM)#did#not#have#a#
strong#influence#on#the#reaction#kinetics#suggesting#that#indeed,#the#quaternary#complex#is#formed#quantitatively#under#these#conditions#
(Fig.#S3).#
#
<5=#*&.GF#Top:#RNA#sequences#used#and#alignment#of#the#PNA#probes#(see#SI#for#explicit#structures);#(A)#/#EI@"#E,.(B)#/#EI@"#G,#and#(C)#/#EI@"#H.in#the#
presence#of#/01EI/01G,#/01E,#/01G,#or#none.#peg#=#[(aminoet h oxy)ethoxy]#acetic#acid,#K#=#l y s ine.#Reaction#condition:#100#nM#of#PNAs#and#100#nM#of#ss#or#
dsRNAs,#30#mM#HEPES-KOH#pH#6.85,#50#mM#NaCl,#5#mM#sodium#ascorbate,#0.02#%#Tween-20;#(D)#Measured#half-life#and#k
app#
of#the#templated#reactions.#
#
#We#then#evaluated#the#performance#of#the#reaction#using#sub-stoichiometric#quantities#of#template#in#order#to#achieve#signal#
amplification.##Using#20%#dsRNA,#the#yield#of#product#exceeded#template#loading#within#less#than#20#min#of#reaction#and#reached#76%#
completion#within#2h#(Fig.#3#A).#Under#these#conditions ,#the#reac tion#reta ined #the#sa m e#disc rimin ation #for#ds RN A#ov er#either#o f#the#ssR NA .##
Reducing#the#concentration#of#dsRNA#to#5#nM#(0.02#equivalent#of#template)#and#0.5#nM#(0.002#equivalent#of#template)#still#afforded#
reaction#discernable#over#backgrou nd .##Run ning #the#rea ction #for#7h #affo rded #a#total#conversion#of#68#%#conversion#with#0.02#equivalent#of#
the#template.#Adjusting#for#the#background#reaction,#this#corresponds#a#19-fold#signal#amplification#(37#%#yield,#19#turnovers).##The#
reaction#with#0.5#nM#template #afford ed #3#%#yield#(adjusting#for#the#background#conversion)#after#7h,#representing#15-fold#signal#
amplification.##It#should#be#noted#that#the#rate#of#triplex#formation#(10
3
#to#10
4
#M
-1
s
-1
)
[33]
#is#known#to#be#slow e r#th an #h yb rid iza tio n#o f#a #
duplex#(10
6
#M
-1
s
-1
).
[32]
#We#have#recently#shown#that#templated#reactions#engineered#to#yield##a#product#with#lower#affinity#for#the#template#
enhances#the#turnover#of#the#reaction,#provided#reagent#dissociation#is#rate-limiting.
[34]
##However,#in#the#present#case,#the#reaction#is#the#
rate-limiting#step.###
#
#

4
#
<5=#*&.HF#(A)#Plot#of#the#conversion#for#the#templated#reaction#of#@"#H#(250#nM)#with#1#eq.#(250#nM,#black#line)#or#0.2#e q.#of#/#E#(50#nM,#red#line)#in#the#
presence#of#0.2#eq.#(50#nM)#of#dsRNA#(/01EI/01G)#or#ssRNA;#(B)#Plot#of#the#conversion#for#the#reaction#at#various#template#(dsRNA:#/01EI/01G)#loading;#(C)#
Calculated#yield#and#turnovers#for#the#reactions#(the#yield#was#obtained#by#subtracting#the#conversion#observed#in#the#presence#and#absence#of#template).#
Reaction#condition:#30#mM#HEPES-KOH#pH#6.85,#50#mM#NaCl,#5#mM#sodium#ascorbate,#0.02#%#Tween-20.#Yields#are#calculated#based#on#a#titration#of#the#
coumarin,#see#SI#for#details.#
The#number#of#M#nucleobases#in#a#given#PNA#probe#dicta te s#th e #nu m b e r#o f#ca tio nic #ch arg es #an d #th is#h as #a#st ron g #im p ac t#o n #th e#
overall#affinity#and#kinetics#of#the#triplex#formation;#in#particular#at#higher#NaCl#concentrations.
[33]
#The#ruthenium##photocatalyst#further#
adds#two#cationic#charges#to#the#PNA#probes.##Analysis#of#the#sequences#used#in#the#reactions#shown#above#revealed#that#the#/#E#
sequence#(7#charges)#was#overall#more#cationic#than#the#@"#E-H#sequences#(3-4#charges).###By#switching#the#position#of#the#photocatalyst#
and#pyridinium-coumarin,#we#would#alter#the#charge#balance#without#changing#the#overall#PNA#sequ ence s#.#Thus#/#G#and#@"#J.probes#
were#prepared#and#their#performance#in#templated#reactions#studied#(Fig.#4).##Interestingly,#the#reaction#was#found#to#be#more#resilient#to#
high#salt#concentration#and#still#afforded#signal#at#150#mM#NaCl.##Under#the#same#conditions,#/#E#and#@"#H#(same#PNA#sequences#bu t#
opposite#position#of#photocatalyst#and#pyridinium-coumarin#conjugate)#did#not#afford#reaction#pointing#to#the#importance#of#the#overall#
cationic#charges#for#the#triplex#formation#at#high#salt#concentrations.#The#reaction#was#specific#for#the#sequence#of#dsRNA,#a#misma tch ed#
sequence#was#comparable#to#no#template#(Fig.#4B).###Increasin g#th e#le ng th #of#th e#PE G #linke r#(from#9#atoms#to#18#atoms)#betwe en #th e#PN A #
and#reagents#(ruthenium#photocatalyst#and#pyridinium-coumarin#conjugate)#did#not#have#a#significant#impact#on#the#reaction#kinetics#(Fig.#
S4A).##PNAs#modified#at#the#γ#position(L#stereochemistry)#have#been#reported#to#enhance#duplex#stability#and#indu ce#a#helical#
preorganization#of#PNAs.
[35]
##Using#a#γ-modified#PNA#with#serine#side#chains,
[36]
#we#tested#the#reaction#with#the#same#sequence#as#/#G#and#
@"#J#wherein#four#and#three#of#the#positions,#respectively,##contained#a#γ-modification.###Templated#reaction#using#dsRNA#showed#slower#
reaction#for#the#probes#with#γ-modified#PNAs#suggesting#that#such#modifications#are#detrimental#to#the#triplex#formation#(Fig.#S4B).###
#
<5=#*&.JF#(A)#Templated#reaction#of#/#GI@"#J#in#the#presence#of#/01EI/01G.with#different#concentration#of#NaCl#(background#signal#is#from#ssRNA#or#none);#
(B)#templated#reaction#of#/#GI@"#J#in#the#presence#of#fully#matched #ds RN A#(/01EI/01G)#or#mismatched#dsRN A#sequen ce#(/01HI/01J)#in#100#mM#NaCl#
concentration.#Reaction#condition:#100nM#of#PNAs#and#100#nM#of#ss#or#dsRNAs,#30#mM#pH#6.85#HEPES-KOH,#5#mM#sodium#ascorbate,#0.02#%#Tween-20,#
incubation#time:#30#min.#
'
Detection'of'pre-miR-31'sequence'using'dsRNA-templated 'rea ction'
Based#on#the#successful#design#of#templated#reactions#responding#to#dsRNA,#we#turned#our#attention#to#the#application#of#this#technology#
for#the#detection#of#pre-microRNA#sequences,#an#important#class#of#ncRN A.#Pre-microRNAs#are#well-known#to#regulate#expression,#serving#

Frequently asked questions

Q1. What have the authors contributed in "Double-stranded rna-specific templated reaction with triplex forming pna" ?

Herein the authors report double‐stranded RNA‐specific templated reaction resulting from PNA‐reagent conjugates that are brought within reactive distance through the formation of sequence‐specific triplexes onto double‐stranded RNA. 

Q2. What is the effect of a templated reaction on the kinetics of ds?

The authors have recently shown that templated reactions engineered to yield a product with lower affinity for the template enhances the turnover of the reaction, provided reagent dissociation is rate-limiting.[34] 

Q3. What is the effect of the modification on the dsRNA?

While γ-modified PNAs have been shown to enhance duplex stability, this modification was found to be detrimental to triplex formation. 

Q4. How many nM of a target sample did the authors detect?

Even at 12.5 nM of a target sample, a distinguishable signal was obtained after 30 min, which implies practical detection of pre-miR-31 at low nanomolar concentration. 

Q5. What are the main considerations for the design of the probes?

The design of the probes was made with the following considerations: each probe should use a different strand of the duplex to minimize any background reaction arising from ssRNA-templated reaction; sequences rich in M and T monomers will form more stable triplex; 8-mer probes should achieve the necessary affinity to yield a templated reaction at low concentration (less than100 nM); the reagents should be separated from the PNA with a short polyethylene glycol spacer (PEG: 9 atoms) to relieve any unfavorable conformational bias. 

Q6. What is the role of the position in the PNAs?

PNAs modified at the γ position(L stereochemistry) have been reported to enhance duplex stability and induce a helical preorganization of PNAs.[35] 

Q7. What is the way to detect ssRNA?

An important design consideration to achieve high discrimination between dsRNA and ssRNA is the length of the PNA probe and the number of M nucleobases. 

Q8. What is the scope of triplex forming PNA?

Rozner and coworkers extended the scope of triplex forming PNA with the introduction of monomers bearing novel nucleobases that extend the Hoogsteen triplex basepairing[26] to any sequence permutation (M•G-C, P•C-G, E•U-A, Fig. 1) and showed that triplex formation was selective for dsRNA over dsDNA.[27-30] 

Q9. What is the role of dsRNA in the detection of pre-miR-?

Detection of pre-miR-31 sequence using dsRNA-templated reactionBased on the successful design of templated reactions responding to dsRNA, the authors turned their attention to the application of this technology for the detection of pre-microRNA sequences, an important class of ncRNA. 

Q10. What is the difference between dsRNA and ssRNA?

It is noteworthy that the high selectivity for dsRNA vs single strand RNA templated reaction is dependent on the salt concentration; in the absence of NaCl, the reaction of dsRNA was only 2-fold faster than ssRNA (see Fig. S1, A).