Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting

Article abstract of DOI:10.1039/c9ob00885c

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting. With cellular imaging in mind we show that 2′-OMe RNA probes with TO at the 5-position of uracil or the 2′-position of the ribose sugar are particularly effective, exhibiting up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability. Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favourable properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a cellular context.

Full text of DOI:10.1039/c9ob00885c

Organic &
Biomolecular Chemistry
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Design of thiazole orange oligonucleotide probes
for detection of DNA and RNA by fluorescence and
duplex melting†
Cite this: DOI: 10.1039/c9ob00885c
a
a,b
c
a,d
Piotr Klimkowski, Sara De Ornellas, Daniel Singleton, Afaf H. El-Sagheer
a
and Tom Brown
*
We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid
detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of
duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the
linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide
(
major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quino-
line moiety). This information can be used to design probes for detection of target nucleic acids by
fluorescence or duplex melting. With cellular imaging in mind we show that 2’-OMe RNA probes with
TO at the 5-position of uracil or the 2’-position of the ribose sugar are particularly effective, exhibit-
ing up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability.
Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the
TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favour-
able properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a
cellular context.
Received 17th April 2019,
Accepted 3rd May 2019
DOI: 10.1039/c9ob00885c
rsc.li/obc
oped in which the quantum yield increases from 0.02 to 0.23
Introduction
1
3,14
upon target binding,
and ECHO probes, containing thymi-
Thiazole orange (TO) is an unsymmetrical cyanine dye which dine modified with two TO units, give low background in the
can be conjugated to oligonucleotides (ONs) to create fluoro- single stranded form and quantum yields up to 0.44 while
1
–3
15
genic hybridisation probes.
TO becomes highly fluorescent bound to matched RNA targets.
Investigations on the
upon binding to nucleic acids due to restriction of rotation relationship between the fluorogenic properties of thiazole
about the methine bridge, hence TO–ON conjugates have been orange and probe structure have been carried out previously.
4
–10
used for the detection of target nucleic acids.
An important In a study in which TO was linked to the DNA phosphodiester
property of TO–ON probes is the significant increase in duplex backbone via its quinoline moiety, differences in fluorescence
1
1
stability imparted by intercalation of the TO moiety.
A
(F /F ) were moderate; 2.4-fold for the TO-minor groove
ds ss
number of different designs of TO probe systems have been isomer and 0.8-fold the major groove isomer. Attachment of
studied (Table S13†), for example PNA-based forced intercala- TO via its benzothiazole moiety in the major groove gave a 2.7-
tion (FIT) probes containing TO have been shown to produce fold increase in fluorescence upon duplex formation and 1.3-
7
quantum yields (Φ) up to 0.32 on binding to complementary fold increase when directed into minor groove. In a study on
3
,12
nucleic acids.
A DNA version of FIT probes has been devel- PNA in which TO was employed as a surrogate base the fluo-
rescence increase upon duplex formation was large (26-fold)
when TO was attached by its quinoline moiety, but was small
1
6
when it was attached by its benzothiazole moiety.
a
Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford,
OX1 3TA, UK. E-mail: tom.brown@chem.ox.ac.uk
Recently we described a novel fluorogenic probe system
(combination probes) that contains a nucleotide modified
b
Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headley Way,
1
7
Oxford, OX3 9DS, UK
with both TO and a FRET-compatible second dye. This study,
along with an awareness of the numerous potential diagnostic
applications, prompted us to examine the properties of thia-
zole orange oligonucleotides in more detail with the aim of
developing effective TO-probes that are simple to synthesise.
c
ATDBio, School of Chemistry University of Southampton, SO17 1BJ, UK
d
Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum
and Mining Engineering, Suez University, Suez 43721, Egypt
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob00885c
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Material and methods
TO-NHS ester labelling (method 1)
To a solution of thiazole orange TOQ/B6 (20 eq.) in DMF
1
8,19
20
(120 µL), DSC
or PyBop (20 eq.) and NMM (60 eq.) were
added and the mixture was shaken for 30 min at 30 °C (DSC)
or 10 min at 37 °C (PyBOP) at 400 rpm. The mixture was added
to an equal volume of the amino-modified oligonucleotide
(
200 nmol, 1.0 eq.) dissolved in carbonate buffer (NaHCO
3
/
Na CO , 0.5 M, pH = 8.75) in an Eppendorf tube and shaken
2
3
for 4 h at 37 °C. The mixture was desalted by NAP gel filtration
column and purified by RP-HPLC to obtain desired pure
labelled oligonucleotide, isolated yields with DSC were
4
0–50%, and with PyBop were 50–70%. Preparation of the
amino-modified oligonucleotides is described in the ESI† and
a summary of all oligonucleotide sequences is in Tables S1
and S2.†
Fig. 1 Structures of TO-modified nucleotides AE, C6, PA and the TO
moieties used as carboxylic acids or NHS esters for oligonucleotide
labelling.
PyBOP was found to give higher coupling efficiencies (con-
version calculated by crude HPLC chromatograms at 260 nm
after gel filtration, Fig. S2†) and higher isolated yields (amount
of nmol obtained after HPLC purification) than DSC.
duplexes PA and C6 locate the TO dye in the vicinity of the
2
4
major groove, while AE positions it in the minor groove
2
5
TO-NHS ester labelling (method 2)
region, thereby providing structural diversity.
The phosphoramidite monomers of PA and AE, were syn-
A solution of thiazole orange TOQ/B6 NHS ester (5 eq.) in DMF
2
6–28
thesised according to published procedures,
and C6 phos-
(
80 µL) was added to an oligonucleotide (200 nmol) dissolved
in carbonate buffer (NaHCO /Na CO , 0.5 M, pH = 8.75,
20 µL) and shaken for 5 min at 20 °C. The mixture was
phoramidite (amino-C6-dT) was purchased from Glen
Research. All three monomers were incorporated into a series
of 10-mer oligonucleotides (ONs), which were post-syntheti-
cally labelled with TOB6 or TOQ6 in high yield using (benzotria-
zol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
3
2
3
1
desalted by NAP gel filtration column and purified by
RP-HPLC to obtain the desired labelled oligonucleotide.
Complete conversion to the product was observed by HPLC,
and isolated yields were 60–75% (Fig. S3†).
2
9
(
PyBOP) and N-methylmorpholine (NMM) in DMF. PyBOP
was found to give superior yields to previously reported coup-
ling methods which employed N,N′-disuccinimidyl carbonate
3
0,31
(
DSC), the reaction being complete in 4 hours (Fig. S2†).
Results and discussion
Thiazole orange probe design and synthesis
An optimised labelling procedure using the NHS esters of
TOB6 or TOQ6 remarkably gave almost 100% conversion to
Detection and imaging of cellular DNA and RNA are increas- product in just 5 minutes (Fig. S3†). These highly efficient
2
1,22
ingly important fields of study
and fluorogenic intercala- coupling strategies were also successful in a more challenging
tors are potentially valuable in this context. Hence we under- case; the synthesis of probes with double additions of TO
took investigations on the fluorescent and duplex stabilising (Table S2†).
properties of a number of TO probe designs. For synthetic con-
venience we focused on attachment of thiazole orange to the
DNA duplex stabilisation by TO-ODN probes
thymidine nucleotide, but linking it to other nucleotides To identify the effects of base stacking on TO fluorescence and
should also be feasible. ON probes can be modified with TO at duplex stabilisation, four different ODNs sequences were syn-
their nucleobase, ribose sugar or the phosphodiester back- thesised with varied base sequence around the TO-modified
2
3
bone. Another key variable is the position on thiazole orange nucleotide (Table 1A, Table S3†). Addition of TO increased
where the linker between TO and the probe is attached; this duplex stability (melting temperature, T ) to quite varying
m
2
,7
can be via its benzothiazole or quinoline nitrogen. In order degrees when the ODNs were hybridised to fully complemen-
to investigate the effects of these alternative modes of attach- tary DNA strands (ΔT = +1.3 to 14.6 °C) as previously reported
ment on fluorescence and duplex stability, three nucleoside in different probe systems.
monomers, 5-propargylamino-dT (PA), amino-C6-dT (C6) and tion of the thymine nucleobase), the TOB6 modification is
′-aminoethoxy-T (AE) were used to introduce TO into oligonu- more stabilising and the short linker (PA) is more effective
m
4
,32
In the major groove (the 5-posi-
2
cleotides by labelling their aliphatic amino groups (Fig. 1). To than the long linker (C6). In the minor groove (2′-position of
achieve this, thiazole orange was functionalised with a hexa- the sugar, AE) TOB6 and TOQ6 have similar stabilising effects.
noic or decanoic acid linker on either the benzothiazole (TOB6
/
m
In general, a combination of PA with TOB6 gave the best ΔT .
TOB10) or the quinoline moiety (TOQ6/TOQ10). In nucleic acid There are some strong sequence-dependent effects. Most
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Table 1 Changes in melting temperature (ΔT
m
) for DNA duplexes formed by TO-labelled ODN1–4 (A) and changes in fluorescence emission (Fds
, Fss, Fds were obtained in a buffer containing 10 mM phosphate
studies were 3.0 µM of ODN1–4 and 3.3 µM of target strand. For Fds/Fss 0.25 µM of ODN1–4
values are an average of 4 measurements, fluorescence data were measured at least as a duplicate. TOQ/B6: λex
484 nm, λem = 510 nm, slit ex = 7 nm, slit em = 7 nm at 20 °C
/
F
ss) before and after formation of matched and mismatched DNA duplexes (B). T
m
200 mM NaCl at pH 7.0. DNA concentrations for T
m
and 0.28 µM of target were used. T
m
=
1A
ODN1
dGCATX
ODN2
dGCAAX
ODN3
dGCACX
ODN4
Sequence
Duplex
̲
̲
̲
dGCAGX ̲G TACG
ΔT
m
Average
ΔT
Average
ΔT m–mm
c
c
c
c
c
X =
ΔT
m
ΔT
m
ΔT
m
ΔT
m
m–mm
m
m
a
PA-TOQ6
Match
1.3
2.7
6.6
9.0
15.7
6.0
9.8
12.8
14.6
10.5
7.1
9.4
14.6
15.1
11.5
6.3
10.5
12.3
14.6
12.5
5.2
8.1
11.6
13.3
12.6
13.7
b
b
Mismatch
a
PA-TOB6
Match
14.7
12.5
13.3
14.4
Mismatch
a
AE-TOQ6
AE-TOB6
Match
7.7
12.9
6.3
11.9
11.2
10.8
12.3
11.3
12.7
12.8
12.9
9.3
14.5
7.9
8.6
9.5
13.5
8.9
12.7
12.0
9.3
13.3
8.6
11.5
11.6
b
b
Mismatch
a
Match
10.3
Mismatch
12.2
11.4
13.6
12.5
a
C6-TOQ6
C6-TOB6
Match
2.3
11.8
8.4
7.6
4.8
13.5
9.9
5.6
4.7
18.0
11.5
15.6
0.5
9.7
4.0
15.3
10.3
15.0
5.4
4.0
14.7
10.0
14.2
4.8
b
b
Mismatch
a
Match
13.3
10.4
12.0
11.4
Mismatch
12.2
13.8
Average values
Match
Mismatch
5.4
10.1
12.4
17.1
9.3
12.8
10.8
14.3
9.2
14.0
9.0
8.6
13.8
11.5
16.7
T
m
of T*
Match
Mismatch
41.6
24.5
39.6
25.3
47.6
33.8
13.8
49.1
32.4
1B
ODN1
ODN2
dGCAAX
ODN3
dGCACX
ODN4
Sequence
Duplex
dGCATX
̲
̲
̲
dGCAGX ̲G TACG
Average
Average
I /Imm
m
d
e
e
e
e
X =
Fds/Fss
I
m
/Imm
Fds/Fss
I
m
/Imm
Fds/Fss
I
m
/Imm
F
ds/Fss
I
m
/Imm
F
ds/Fss
PA-TOQ6
PA-TOB6
Match
Mismatch
Match
1.0
0.9
3.7
1.1
1.1
3.4
1.4
1.3
5.5
3.8
1.2
1.8
1.5
2.6
5.3
3.3
0.7
1.3
0.9
1.3
2.7
1.3
0.9
2.1
1.2
1.5
4.3
2.4
1.0
2.1
Mismatch
AE-TOQ6
AE-TOB6
Match
Mismatch
Match
2.8
1.5
0.8
0.9
2.0
0.9
7.4
3.2
1.0
1.2
2.7
0.8
4.1
1.5
1.9
1.3
3.5
1.4
1.3
0.6
0.8
0.6
2.1
1.5
3.9
1.7
1.1
1.0
2.6
1.1
Mismatch
C6-TOQ6
C6-TOB6
Match
Mismatch
Match
1.0
0.5
6.6
2.3
1.5
3.3
3.7
2.5
8.6
5.5
1.3
1.9
3.1
0.9
6.9
2.6
3.4
2.8
1.4
0.6
3.0
1.1
2.1
3.8
2.3
1.1
6.3
2.9
2.1
3.0
Mismatch
Average values
Match
mismatch
2.7
1.2
2.0
4.6
2.9
1.6
3.8
2.0
2.2
1.7
0.9
2.1
a
b
c
In comparison to the matched unmodified duplex. In comparison to the mismatched unmodified duplex. Difference between T
m
of matched
d
(
m) and mismatched (mm) target in °C, * = T
sity for Fds and Fss of ODN probes with matched or mismatched targets. Ratio of fluorescence intensity of matched (I
intensity of probes-targets duplexes at λem, max
m
value of unmodified matched and mismatched duplexes. Ratio of integrated fluorescence inten-
e
m
) and mismatched (Imm)
.
notably, in fully matched DNA duplexes, placing the TO-modi- The TO-modified ODNs were also evaluated in mismatched
fied nucleotide between thymine bases (TXT) gives the least duplexes by replacing the adenine base opposite to the TO-
stable duplexes, whereas the other stacking environments are modified T with thymine to create a T : T mismatch directly
significantly more stabilising and are similar to each other. opposite the TO-T nucleotide. As expected, melting tempera-
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tures were significantly lower than those with the fully comp- Table 2 Changes in melting temperature (ΔT
m
) ODNs against RNA
targets and 2’-OMe–(ORNs) against DNA and RNA targets. Conditions:
lementary strands, PA-TOB6 showing the greatest mismatch
discrimination of the series, and the TXT sequence (ODN1)
was the most sensitive to the mismatch. This study indicates
that thiazole orange does not prevent thermal mismatch dis-
crimination, although it does reduce it relative to unmodified
mismatched duplexes. However, locating the mispaired base
see Table 1
X = AE
X = C6
X = T/U
Target
TOQ6 TOB6 TOQ6 TOB6
—
T
m
a
m
Probe
(matched) ΔT
b
b
b
b
directly opposite thiazole orange is not the optimum strategy ODN1
RNA
5.7
—
—
—
—
3.3
1.3
0.6
0.8
8.2
5.2
5.9
6.3
35.9
32.0
48.9
44.6
b
b
b
ODN2
—
—
—
for detection of point mutations or single nucleotide poly-
morphisms (SNPs). A superior strategy is discussed later.
ODN3
ODN4
Fluorescence properties of TO-ODN probes with DNA targets
2′-OMe–(ORN1)
′-OMe–(5′-
GCAU UUACG)
RNA
DNA
7.8
7.7
4.7
7.4
14.3 25.2
42.6
2
18.0 19.3 9.7
Fluorescence emission spectroscopy of the TO-modified ODNs
as single strands and with their matched and mismatched
complementary DNA strands showed clear trends (Table 1B,
2
2
′-OMe–(ORN2)
′-OMe–(5′-
RNA
DNA
6.2
10.3 8.8
4.9
0.2
4.3
0.7
6.8
44.6
35.8
Fig. S5†).‡ Both the position and mode of attachment of the GCAA AUACG)
dye strongly influence the fluorescence properties. C6-TOB6
2′-OMe–(ORN3)
and PA-TOB6 provided good discrimination between single 2′-OMe–(5′-
RNA
DNA
4.0
4.8
0.5
4.6
12.7 43.2
60.1
11.1 15.0 8.0
GCAC CUACG)
strand (ss) and double strand (ds), and C6 is better than PA
with an average 6.3-fold increase in fluorescence on duplex for- 2′-OMe–(ORN4)
RNA
DNA
6.5
8.4
5.3
8.5
0.4
3.7
3.9
9.7
58.9
49.1
2
′-OMe–(5′-
mation. Fluorescence-based mismatch discrimination of
TO-ODNs (1–4) against DNA targets was best achieved by PA
and C6 attached to TOB6, i.e. with attachment of thiazole
orange in the major groove. In contrast, for AE-TO; when the
GCAG GUACG)
a
b
In comparison to the unmodified duplex. No data obtained.
dye is situated in the minor groove, the greatest changes in
fluorescence occur for attachment via the quinoline moiety endogenous RNase H.
1
3,36
2′-OMe probes have been used pre-
(
TOQ6) (Fig. S5, Table S8†). In terms of the base stacking viously in combination with TO using a different design of the
3
7
environment, the weakest fluorescence enhancement was TO monomer. In order to elucidate the effects of the 2′-OMe
observed with TO adjacent to guanine bases (ODN4) as pre- modification on the properties of TO probes, 2′-OMe probes
1
8,33
viously reported.
The reason for this is not because the TO (2′-OMe–(ORN1–4)) with the same sequences as ODN1–4, con-
moiety is less fluorescent (i.e. strongly quenched) in this taining AE or C6 modifications, were synthesised and labelled
environment, but rather that the single stranded probe is with TOQ6 and TOB6. These probes were tested against both
poorly quenched (Fig. S5, Tables S8 and S10†). This could be DNA and RNA targets (Table 2). Increases in melting tempera-
due to strong single stranded stacking between TO and ture were greater against DNA than RNA, and the minor groove
guanine bases. Attempts to reduce single stranded fluorescence (AE) modification was superior to the major groove (C6). For
3
4
(
F
ss) of ODN4 by replacing guanine with inosine resulted in AE there were no great differences between benzothiazole and
decreased Fss but unfortunately sequence specificity and duplex quinoline attachment (Table 2, Fig. S15 and S16†). The highest
stabilisation were compromised (Fig. S19 and S20†).
increases in T were observed for 2′-OMe–(ORN1) with DNA
m
targets (AE-TOQ6 +18.0 °C and AE-TOB6 +19.3 °C). These
increases are quite remarkable for a single intercalating fluoro-
phore, and double labelled 2 × AE-TOQ6 provided even greater
Duplex stabilisation by 2′-O-methyl–RNA TO (2′-OMe–(ORNs))
probes with DNA and RNA targets
duplex stabilisation (ΔT
m
= + 29.1 °C, Table S4†). Probes of
An important application of fluorogenic oligonucleotide
probes is detection of DNA and RNA in both live and fixed
cells. A common approach is to use 2′-O-methyl oligoribonu-
cleotide probes (2′-OMe-ORN) instead of DNA probes, as the
former are much more stable in live cells, and the latter can
also cause degradation of the target mRNA via the action of
this design with multiple TO insertions have great potential in
duplex stabilisation and importantly are simple to synthesise.
It is significant that the ability of 2′-OMe–(ORN1–4) to dis-
criminate matched from mismatched DNA targets was pre-
served, and on average ΔTm was improved slightly in compari-
son to ODNs probes.
35
Next we examined the effect of mismatch position on ΔT
i.e. Tm of duplex containing all Watson–Crick base pairs – Tm
m
(
‡
In this study the ratio of double to single stranded fluorescence (Fds/Fss) was of duplex containing a single mismatch) by constructing five
calculated on the basis of area under the fluorescence emission spectra from
different DNA hybrids that gave a single mismatch against
TO-2′-OMe–(ORN3) in various positions from −2 (2 bases to
the 5′-side of TO) to +2 (2 bases to the 3′-side of TO) (Fig. 2,
Table S7†). A similar study was performed by Okamoto with
5
10 nm to 650 nm. More favourable Fds/Fss values are obtained if a narrower
band is selected, e.g. For ODN3 C6-TOB6 against its complementary DNA target
between 510 nm and 555 nm Fds/Fss = 7.7 compared to 6.9 in the region 510 nm
to 650 nm, and in case of 2′-OMe–(ORN3) AE-TOQ6 against its complementary
DNA target (discussed later) the relevant values are 43.9 compared to 33.9.
3
8
TO probes of a different chemical composition. For both
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better than the equivalent deoxyribose (DNA) versions. The
probes showed the same structural preference for dye orien-
tation as the DNA probes; the most impressive fluorescence
enhancement was observed for AE-TOQ6 and C6-TOB6 against
DNA targets. Limited duplex fluorescence was observed when
the dye was attached in the alternative orientation (AE-TOB6 or
C6-TOQ6, Fig. 3, Table 3). Surprisingly in one case (AE-TOB6 in
2
′-OMe–ORN4) the single stranded oligonucleotide was four
times as fluorescent as its duplex with complementary DNA,
and twice as fluorescent as its duplex with RNA. This sequence
2
′-OMe–(5′-GCAG ̲X ̲G ̲U ACG) places the thiazole orange between
guanine bases and it is likely that the resulting stacking inter-
actions are sufficiently strong to allow the dye molecule to
adopt a planar fluorescent conformation. The same effect is
observed with the equivalent DNA probe (AE-TOB6 in ODN4,
Fig. 2 UV melting for 2’-OMe–(ORN3) (2’-OMe–(5’-GCAC CUACG))
TO-modified oligonucleotides probes against various DNA targets.
Position of mismatch refers to a mismatch in the target strand counting Fig. S12†) but to a smaller extent, and it is unclear why in this
from the TO-modified base, e.g.: position “0” is directly opposite modifi- single case the 2′-O-methyl sugars promote fluorescence in the
cation and “−2” is two nucleobases towards 5’ end of the probe
single strand whereas in other cases they suppress it.
m m
sequence. Number above bar = ΔT to match target, T for fully
Nevertheless this result shows the importance of evaluating
different modes of attachment between TO and DNA/RNA. The
C6-TOB6 modification in 2′-O-methyl RNA gave good Fds/Fss
ratios with DNA targets, for example 2′-OMe–(ORN3) and 2′-
OMe–(ORN1) gave fluorescence enhancements of 17-fold and
matched targets 2’-OMe–(ORN3) AE-TOQ6: 51.7 °C; 2’-OMe–(ORN3)
C6-TOB6: 52.5 °C, all samples measured at 1.0 µM with 1.1 eq. of target.
Mismatch sequences see: Table S1,† ODN3 – targets from −2 to +2.
AE-TOQ6 and C6-TOB6 mismatch discrimination at positions 15-fold respectively (Fig. 4 and S22†). Somewhat smaller
1 and +1 was excellent (ΔT > 20 °C) and far superior to the increases were observed with RNA targets. With complemen-
case when the mispaired base is directly opposite TO (ΔTm
tary DNA targets, the AE-TOQ6 modification attached to 2′-
–12 °C). This position-dependence of mismatch discrimi- OMe–(ORN3) gave an impressive 34-fold enhancement (Fig. 4)
nation is a critical consideration when designing TO probes to (44-fold if calculated in the 510–555 nm range), and when
−
m
=
9
detect mutations or SNPs.
attached to 2′-OMe–(ORN1) the enhancement was 22-fold
27-fold for the 510–555 nm range). For double TO-labelled
probes, Fds/Fss of 2 × AE-TOQ6 2′-OMe–(ORN1) was reduced to
4-fold (Table S9†) which may be due to mutual quenching of
(
Fluorescence properties of 2′-OMe–(ORNs) probes against DNA
and RNA targets
1
1
4
The 2′-OMe–(ORN1–4) series of probes show great potential as TO dyes in the double strand. However, we have only so far
fluorogenic sensors of complementary DNA and RNA. The studied a single example of dual-labelled TO probes, and it
fluorescence enhancement upon duplex formation for may be possible to optimise the design to improve their fluo-
AE-TOQ6 and C6-TOB6 is excellent, and in the best cases much rescent properties.
Fig. 3 Fluorescence emission intensities at λem, max of a single stranded 2’-OMe–(ORN1–4) probes and DNA and RNA matched targets duplexes.
Conditions: see Table 1.
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Table 3 Changes in fluorescence emission (Fds/Fss) for 2’-OMe–(ORN1–4) with DNA targets before and after formation of matched and mis-
matched duplexes. Conditions: see Table 1. In this study Fds/Fss was calculated on the basis of area under the fluorescence emission spectra from
5
10 nm to 650 nm. More favourable values are obtained if a narrower band in the emission spectra is selected, e.g. For 2’-OMe–(ORN3) AE-TOQ6
against its complementary DNA target between 510 nm and 555 nm; Fds/Fss = 43.9 compared to 33.9 integrated in the region from 510 nm to
50 nm
6
2
′-OMe–(ORN1)
2′-OMe–(ORN2)
2′-OMe–(ORN3)
2′-OMe–(ORN4)
rGCAGXGUACG
Average
Average
Sequence
rGCAUX
̲
rGCAAX
̲
rGCACX
̲
̲
a
b
a
b
a
b
a
b
X =
Duplex
Fds/Fs s
I
m
/Imm
Fds/Fss
I
m
/Imm
F
ds/Fss
I
m
/Imm
Fds/Fss
I
m
/Imm
Fds/Fss
m
I /Imm
AE-TOQ6
Match
Mismatch
Match
21.5
8.9
1.3
1.6
2.8
0.8
7.6
3.0
0.9
1.6
2.6
0.4
33.9
17.0
3.5
2.2
1.3
3.1
1.4
0.2
0.3
2.1
0.7
16.5
7.6
1.5
1.5
2.4
0.8
AE-TOB6
Mismatch
2.6
C6-TOQ6
C6-TOB6
Match
Mismatch
Match
4.2
1.7
14.6
2.7
2.3
6.0
9.1
2.8
17.5
8.6
3.4
1.9
6.4
2.0
17.2
3.9
2.9
3.8
1.0
0.7
3.7
1.2
1.3
3.9
5.2
1.8
13.2
4.1
2.5
3.9
Mismatch
Average values
Match
Mismatch
10.4
3.7
2.4
8.8
4.0
1.7
15.2
6.4
2.1
2.0
0.9
1.6
a
b
Ratio of integrated fluorescence intensity for Fds and Fss of 2′-OMe–(ORN) probe with matched or mismatched target. Ratio of fluorescence
) and mismatched (Imm) intensity at λem, max
intensity of matched (I
m
.
Table 4 Fluorescence quantum yields of ODN1 (5’-GCAT TTACG),
ODN3 (5’-GCAC CTACG), 2’-OMe–(ORN1) (2’-OMe–(5’-GCAU UUACG))
and 2’-OMe–(ORN3) (2’-OMe–(5’-GCAC CUACG)) oligonucleotides
probes with matched DNA and RNA targets. Samples were measured at
λex = 484 nm, λem = 490 nm, ex slit width = 3 nm, em slit width = 3 nm,
20 °C. Reference dye: fluorescein in 0.1 M NaOH
Φ
ss
DNA
RNA
DNA/ss RNA/ss
ODN1
AE-TOQ6 0.089 0.279 0.278
AE-TOB6 0.054 0.067 0.172
C6-TOQ6 0.115 0.118 0.208
C6-TOB6 0.056 0.469 0.477
3.1
1.2
1.0
8.4
3.1
3.2
1.8
8.5
2
′-OMe–(ORN1) AE-TOQ6 0.016 0.580 0.215 36.3
13.4
3.2
6.4
AE-TOB6 0.035 0.065 0.113
C6-TOQ6 0.023 0.152 0.147
1.9
6.6
C6-TOB6 0.019 0.493 0.477 25.9
25.1
Fig. 4 Examples of fluorescence emission spectra of single stranded ODN3
AE-TOQ6 0.051 0.061 0.043
AE-TOB6 0.034 0.053 0.062
C6-TOQ6 0.036 0.045 0.050
C6-TOB6 0.050 0.073 0.063
1.2
1.6
1.3
1.5
0.8
1.8
1.4
1.3
(
ss) probes and DNA or RNA matched target duplexes. Probes: 2’-OMe–
(
ORN3) (5’-GCAC CUACG) were X = AE-TOQ6 or C6-TOB6 with struc-
tures of modification and TO isomer with optimum fluorescence.
Examples of Fds/Fss are calculated in range 510–555 nm. Conditions: see
Table 1.
2′-OMe–(ORN3) AE-TOQ6 0.013 0.318 0.162 24.6
12.5
2.7
3.1
8.6
AE-TOB6 0.013 0.086 0.035
C6-TOQ6 0.014 0.096 0.044
C6-TOB6 0.022 0.382 0.190 17.3
6.9
7.1
Fluorescence quantum yields of AE-TOQ6 of 2′-OMe–(ORN3)
and 2′-OMe–(ORN1) were 0.318 and 0.580 respectively against
3
9
DNA targets (Table 4). This pleasingly high fluorescence rescence intensity (Imatched/Imismatched) of 2′-O-methyl RNA TO
enhancement is in part due to the low background fluo- probes against DNA targets is significant, e.g. for 2′-OMe–
rescence observed in the single stranded 2′-OMe RNA TO-oligo- (ORN1) and 2′-OMe–(ORN3) with C6-TOB6, 6.0 and 3.8-fold
nucleotides (Φ = 0.013–0.022, 2′-OMe–(ORN1) and 2′-OMe– differences respectively were observed, whereas for ODN1 and
(ORN3)) in comparison to equivalent ODN probes (Φ = ODN3 probes, the corresponding differences were 3.3 and 2.8-
0
.050–0.089, Table 4). This puts our simplified design on par fold. Varying the mismatch position relative to thiazole orange
1
3,15
with other TO probe systems.
If a mispaired base is posi- in the 2′-OMe–(ORN3) sequence gave significant fluorescence
tioned opposite to TO, discrimination on the basis of fluo- discrimination at positions −1 and +1 (3.4 to 3.6-fold for C6-
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TOB6 and 2.0 to 2.4 for AE-TOQ6), i.e. the positions at which investigated. For both AE-TOQ6 and C6-TOB6 modifications, as
1
8
mismatch discrimination by Tm is maximum (Fig. S21†). With previously described in different systems, mismatch dis-
further development, the favourable fluorescence properties of crimination against DNA targets at positions −1 and +1 was
these simple 2′-OMe TO probes may prove useful in detection excellent and far superior to the case when the mispaired base
of DNA in cells.
is directly opposite TO. This is a crucial consideration when
designing TO probes to detect mutations or SNPs. Similar
effects were observed previously in other TO probe configur-
Effect of linker length on the properties of 2′-OMe–(ORN1)
AE-TO probes
3
8
ations. The fluorogenic properties of DNA TO-probes are
limited by residual fluorescence in the single stranded state
due to the cationic TO interacting with anionic DNA, causing
single stranded TO-probes to adopt stable secondary structures
in which the dye is fluorescent. This has been addressed pre-
viously by the use of PNA probes and ECHO probes. Here we
confirm that 2′-O-Me RNA probes also reduce the background
As shown above, the properties of the TO probes are somewhat
altered when they are attached to the 5-position of the nucleo-
base by different linkers (C6 or PA) presumably due to differ-
ences in linker length. We also explored the effects of the
length of the linker between the TO and the 2′-position of the
sugar (AE modification). For this study TOB10 and TOQ10 NHS
esters were synthesized and used to label the 2′-OMe–(ORN1)
AE and C6 probes (Table S2†). UV melting studies with fully
matched DNA and RNA targets showed the same trends as pre-
viously observed for AE and C6-TOB6/Q6 with slightly lower
1
5
fluorescence whilst maintaining simplicity of design and
convenience of synthesis. Anomalous fluorescence results were
obtained when TO in an AE-TOQ6 context is placed between
two guanine bases and this highlights the importance of
studying the system in detail. In conclusion, the 2′-OMe-RNA
TO-probes presented here have potential for applications in
cell imaging of DNA and RNA and warrant further investi-
gation, particularly in FRET systems in combination with
other fluorophores.
m
increases in T (Fig. S18†). The fluorescence changes also fol-
lowed the same trends as the C6 and AE versions against DNA
and RNA targets, but with no advantage over the shorter linker
(Fig. S17†). Fluorescence-based mismatch discrimination was
compromised, and taking the above results into account there
is no advantage to be gained by increasing linker length from
6
to 10 atoms.
Circular dichroism spectroscopy of the modified TO-con-
Conflicts of interest
taining DNA duplexes (TO-ODNs) indicated in general the for-
mation of B-like DNA with no major perturbation from the
unmodified structures (Fig. S27B, S28 and S30†). CD spectra of
the 2′-OMe-(ORN) duplexes are very similar to the equivalent
unmodified duplexes (Fig. S27A, S29 and S31†).
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by the Marie Skłodowska-Curie
Innovative Training Network (ITN) ClickGene (H2020-
MSCA-ITN-2014-642023) and UK BBSRC grant BB/J001694/2
Conclusions
In summary, we have synthesised a range of thiazole orange
(
extending the boundaries of nucleic acid chemistry) and BB/
(TO) probes in which TO is attached to the nucleobase or
R008655/1.
sugar of a thymidine nucleotide using various linkers. The pro-
perties of duplexes between TO-probes and their DNA and
RNA targets depend on the length of the linker between TO
and the oligonucleotide, the position of attachment of thiazole
orange to the nucleotide (nucleobase – major groove or sugar-
minor groove) and the position of attachment of the linker to
thiazole orange (via the benzothiazole or quinoline moiety).
Attachment of thiazole orange via its benzothiazole moiety to
the major groove of oligonucleotides (C6-TOB6) and via the qui-
noline moiety in the minor groove (AE-TOQ6) provides signifi-
cant fluorescence enhancement on duplex formation with
DNA or RNA targets. Duplex melting experiments showed
higher stabilisation by C6-TOB6 than C6-TOQ6 whereas the AE
modification showed similar effects for B6 and Q6 attachment.
Preliminary experiments with double labelled 2′-OMe RNA (2 ×
AE-TOQ6) hybridised to complementary DNA indicate that mul-
tiple additions of TO can provide significantly enhanced
duplex stability. However, the effects of multiple TO insertions
on sequence specificity in a genomic context remain to be
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