Pyrene-functionalized triazole-linked 2′-deoxyuridines - Probes for discrimination of single nucleotide polymorphisms (SNPs)

Article abstract of DOI:10.1039/c0cc01133a

Oligonucleotides modified with pyrene-functionalized triazole-linked 2′-deoxyuridines display remarkable hybridization-induced increases in fluorescence emission and enable efficient fluorescent discrimination of SNPs via G-specific quenching.

Full text of DOI:10.1039/c0cc01133a

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Pyrene-functionalized triazole-linked 20-deoxyuridines—probes for
discrimination of single nucleotide polymorphisms (SNPs)w
Michael E. Østergaard,a Dale C. Guenther,a Pawan Kumar,ab Bharat Baral,a Lee Deobald,c
Andrzej J. Paszczynski,c Pawan K. Sharmab and Patrick J. Hrdlicka*a
Received 27th April 2010, Accepted 18th May 2010
First published as an Advance Article on the web 4th June 2010
DOI: 10.1039/c0cc01133a
Oligonucleotides modified with pyrene-functionalized triazole-
linked 20-deoxyuridines display remarkable hybridization-
induced increases in fluorescence emission and enable efficient
fluorescent discrimination of SNPs via G-specific quenching.
Single nucleotide polymorphisms (SNPs) are the most prevalent
mutation type in the human genome (B10 million SNPs) and
commonly linked to diseases and individual responses to
therapeutics. Development of SNP typing technologies has
accordingly been an area of much recent focus.1 The use of
oligonucleotides (ONs) modified with base discriminating
fluorescent (BDF) nucleotides is a particularly interesting
approach toward this end, as complementary targets are
discriminated from singly base pair mismatched sequences
by differences in fluorescence emission.2 Attachment of polarity-
sensitive fluorophores to the C5-position of 20-deoxyuridines
via alkynyl linkages has been a very successful strategy toward
development of BDFs.3 Interestingly, while the Cu(I) catalyzed
azide alkyne Huisgen 1,3-dipolar cycloaddition4 has been
Scheme 1 Synthesis of monomers X and Y. Reagents and conditions:
(a) sodium ascorbate, CuSO4, THF
: H2O : tBuOH (2X: 52%
extensively utilized in nucleotide chemistry5 to, for example,
introduce moieties at the C5-position of pyrimidines,6 it has
not been utilized to generate BDF nucleotides. Motivated by
this, the general interest in pyrene-modified ONs,7 and our
own interest in C5-functionalized8 and pyrene-functionalized
ONs9 for potential biomedical applications, we set out to
synthesize and study C5-pyrene-functionalized triazole-linked
20-deoxyuridine monomers X and Y (Scheme 1). We surmised
that attachment of pyrene fluorophores to the C5-position
of 20-deoxyuridine via short linkers would lead to precise
positional control of the label and development of new BDF
nucleotides.
C5-Ethynyl 50-O-(4,40-dimethoxytrityl)-20-deoxyuridine 110
is a readily attainable starting material (63% over three steps
from 5-iodo-20-deoxyuridine) for the synthesis of target
amidites 3X/3Y. Coupling of 1 with either 1-azidopyrene11
or 1-azidomethylpyrene12 in a ‘‘click reaction’’ afforded
yield; 2Y: 80% yield), (b) 2-cyanoethyl N,N0-diisopropylchloro-
phosphoramidite, DIPEA, CH2Cl2 (3X: 73% yield; 3Y: 66% yield),
and (c) DNA synthesizer. Pyr = pyren-1-yl.
5-(1,2,3-triazol-4-yl)-20-deoxyuridines 2X and 2Y in 52% and
80% yield, respectively. Subsequent O30-phosphitylation
provided phosphoramidites 3X and 3Y (73% and 66%),z
which were incorporated once into the center of mixed sequence
13-mer ONs3a using automated solid phase DNA synthesis
(0.2 mmol scale). The nucleotides flanking the X/Y monomers
were systematically varied to study the influence of neighboring
nucleotides on biophysical properties. Coupling yields of
498% were observed for unmodified monomers as well as
for phosphoramidites 3X and 3Y (15 min coupling time; DCI
as activator).w The identity and purity of purified ONs was
verified by MALDI-TOF MS analysis (Table S2, ESIw) and
RP-HPLC (480%), respectively.w
Incorporation of monomer X into ONs (ON5–ON8) results
in decreased thermal denaturation temperatures (Tm’s) of the
corresponding duplexes with complementary DNA relative to
reference strands ON1–ON4 (DTm between ꢀ6.5 1C and ꢀ9.0 1C,
Table 1, Fig. S1, ESIw). These results are in agreement
with previous observations where incorporation of bulky
C5-substituted pyrimidine monomers leads to decreased thermal
affinity toward DNA targets.6,13 While reference strands
ON1–ON4 display the expected pattern of discrimination with
singly mismatched DNA targets, ON5–ON8 almost invariably
exhibit poor mismatch discrimination when the mismatched
a Dept. of Chemistry, Univ. of Idaho, Moscow, ID-83844, USA.
E-mail: hrdlicka@uidaho.edu; Fax: +1 208-885-6173;
Tel: +1 208-885-0108
b Dept. of Chemistry, Kurukshetra University, Kurukshetra 136119,
India
c Environmental Biotechnology Institute, Univ. of Idaho, USA
w Electronic supplementary information (ESI) available: General
experimental section, protocols for synthesis, purification and
characterization of nucleosides and ONs (including NMR-spectra and
MS-data), details of thermal denaturation and fluorescence studies,
representative Tm-curves, additional fluorescence emission spectra, data
for studies against RNA targets. See DOI: 10.1039/c0cc01133a
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Table 1 Thermal denaturation temperatures of duplexes between
ON1–ON12 and complementary (bold) or mismatched DNA targetsa
Tm (DTm)b/1C Mismatch DTmc/1C
B = A
50-CG CAA ATA AAC GC 48.5
C
G
T
ON Sequence
1
2
3
4
ꢀ10.0 ꢀ5.0 ꢀ9.0
ꢀ13.5 ꢀ9.5 ꢀ9.0
ꢀ13.0 ꢀ9.5 ꢀ10.0
ꢀ11.0 ꢀ9.0 ꢀ11.0
50-CG CAA CTC AAC GC 55.5
50-CG CAA GTG AAC GC 55.5
50-CG CAA TTT AAC GC 48.5
5
6
7
8
50-CG CAA AXA AAC GC 42.0 (ꢀ6.5)
+1.0 +2.0 ꢀ1.0
ꢀ
ꢀ
50-CG CAA CXC AAC GC 48.5 (ꢀ7.0)
ꢀ1.5
0.0 ꢀ1.5
50-CG CAA GXG AAC GC 47.5 (ꢀ8.0)
0.0 ꢀ10.5 ꢀ3.5
ꢀ
ꢀ
50-CG CAA TXT AAC GC 39.5 (ꢀ9.0)
+0.5 ꢀ1.0 ꢀ1.0
9
50-CG CAA AYA AAC GC 43.5 (ꢀ5.0)
ꢀ5.0 ꢀ4.5 ꢀ5.5
ꢀ11.0 ꢀ6.5 ꢀ6.0
ꢀ5.0 ꢀ10.0 ꢀ7.0
ꢀ6.5 ꢀ5.5 ꢀ7.5
ꢀ
ꢀ
10 50-CG CAA CYC AAC GC 54.5 (ꢀ1.0)
11 50-CG CAA GYG AAC GC 50.0 (ꢀ5.5)
ꢀ
ꢀ
12 50-CG CAA TYT AAC GC 43.5 (ꢀ5.0)
a
Tm’s measured as maximum of first derivative plot of melting curves
(A260 vs. T) recorded in medium salt buffer solution ([Na+] =
110 mM, [Clꢀ] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4), [EDTA] =
0.2 mM) using 1.0 mM concentrations of each strand. Tm’s are an
b
average of at least two measurements. (DTm)
Tm-value relative to unmodified reference duplex e.g. ON5:DNA vs.
= change in
c
ON1:DNA. Mismatch DTm
= difference in Tm-value between
Fig. 1 Steady state fluorescence spectra of single stranded ON7
(upper panel) or ON10 (lower panel) and corresponding duplexes
with matched or mismatched DNA targets (mismatched nucleotide
opposite to modification in parenthesis). Buffers and concentrations
are as described for Tm experiments (see footnote, Table 1); lex = 344 nm;
T = 5 1C.
mismatched duplex and complementary duplex; mismatched
sequences: 30-GC GTT TBT TTG CG-50 (for ON1/ON5/ON9),
30-GC GTT GBG TTG CG-50 (for ON2/ON6/ON10), 30-GC GTT
CBC TTG CG-50 (for ON3/ON7/ON11) and 30-GC GTT ABA TTG
CG-50 (for ON4/ON8/ON12) where B is A, C, G and T.
nucleotides are positioned centrally opposite of monomer X
(compare mismatch DTm-values for ON1 and ON5, Table 1).
nucleotide similar to C5-pyrenecarboxamide-modified propargyl-
linked 20-deoxyuridines,3a which have been used to discriminate
SNP sites in human breast cancer.14 We speculate that the
fluorophore of monomer X is: (a) positioned in the polar
major groove and highly emissive when ONs are hybridized
to complementary targets, and (b) intercalating when ONs are
hybridized to mismatched targets leading to reduced thermal
discrimination15 and variable fluorescence emission that
depends on the nature of nearby nucleotides—guanines are
efficient quenchers of pyrene monomer fluorescence.16 Direct
comparison with C5-pyrenecarboxamide-modified propargyl-
linked 20-deoxyuridines in identical sequence contexts reveals
that SNP discrimination by ON6/ON7 is equally efficient.3a
With the exception of ON12, hybridization of ONs modified
with monomer Y to complementary DNA results in remarkable
increases in fluorescence intensity (9- to 23-fold relative to SSP
at lex = 377 nm, Fig. 1 lower panel, Fig. 2 lower panel and
Fig. S6–S8, ESIw) to give emission spectra with three
sharp peaks with maxima at 377, 397 and 417 nmy (Fig. 1).
Interestingly, brighter fluorescence was observed for these
probes as compared to ON5–ON8 (monomer X).z Very few
singly modified pyrene-labelled ON probes have been reported
to exhibit such levels of hybridization-induced increases in
ONs that are singly modified with monomer
Y
(ON9–ON12) display less pronounced decreases in thermal
affinity toward complementary DNA (DTm between ꢀ1.0 1C
and ꢀ5.5 1C, Table 1, Fig. S2, ESIw) and more efficient
mismatch discrimination than ON5–ON8 (compare mismatch
DTm values for ON1, ON5, and ON9, Table 1). These
differential trends suggest that the subtle change in linker
chemistry between the pyrene and triazole units in monomers
X and Y markedly influences the binding mode of the
C5-substituent.
Next, steady-state fluorescence emission spectra of
ON5–ON12 and the corresponding duplexes with matched
or singly mismatched DNA targets were recorded at 5 1C using
an excitation wavelength of lex = 344 nm. Duplexes between
ON5–ON8 (monomer X) and complementary DNA exhibit
two broad maxima at 382 and 400 nm. Hybridization is
accompanied by substantial increases in fluorescence intensity
at lem = 382 nm (2- to 5-fold, Fig. 1 upper panel, Fig. 2 upper
panel and Fig. S3–S5, ESIw). In contrast, hybridization of
ON6 or ON7 to DNA strands with mismatched nucleotides
opposite of monomer X results in significantly lower fluorescence
output than with matched duplexes (Fig. 1 upper panel
and Fig. 2 upper panel). Flanking nucleotides influence the
fluorescence properties of monomer X as evidenced by the
much more efficient fluorescent discrimination of SNP targets
when monomer X is flanked by cytidines or guanosines
(ON6/ON7) than when flanked by adenosines or thymidines
(ON5/ON8, Fig. 2 and Fig. S3–S5, ESIw). The results
demonstrate that monomer X is a base-discriminating fluorescent
fluorescence intensity,9a,17 rendering monomer
Y as an
interesting building block for diagnostic applications. Unlike
ONs modified with monomer X, ON9–ON12 do not efficiently
discriminate SNP targets except those resulting in Y:G
mismatches (Fig. 1 lower panel, Fig. 2 lower panel and
Fig. S6–S8, ESIw). This, along with thermal denaturation
studies, suggests that the fluorophore of monomer Y is less
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Spectrometry Center and Ms. Brooke A. Anderson (Univ. of
Idaho) for mass spectrometric analyses.
Notes and references
z The identity of the reported compounds was fully ascertained
by NMR (1H, 13C, 31P, COSY, HSQC and/or DEPT) and
MALDI-HRMS, while purity was determined by 1D NMR.
y Single stranded probes and duplexes with mismatched targets generally
result in red-shifting of fluorescence emission peaks by up to 5 nm.
z Relative fluorescence emission quantum yield of ON10:DNA = 0.16.
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Fig. 2 Fluorescence intensity of single stranded probes (SSPs) and
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prone to intercalate upon hybridization to mismatched targets
than that of monomer X. The different binding modes are
related to the subtle change in linker chemistry between Y and
X monomers, but are not fully understood at the molecular
level. Diagnostic assays employing monomer Y will—unlike
assays employing monomer X—require precise temperature
control to minimize undesired duplex formation with non-targets
and concomitant false positives.
Finally, given the importance of probes for detection of
RNA targets to elucidate biological roles of RNA in living
organisms,18 we recorded thermal denaturation curves and
fluorescence emission spectra of the representative probes
ON6 and ON10 and their duplexes with complementary or
singly mismatched RNA targets (Table S3, Fig. S9 and S10,
ESIw). Similar trends were observed for ON6 (monomer X;
efficient SNP discrimination) and ON10 (monomer Y;
pronounced hybridization-induced increases, discrimination of
Y:G mismatches) as when targeting the corresponding DNA
strands, which emphasizes the generality of these probes.
To sum up, two fluorescent nucleotide monomers have been
prepared in five steps from commercially available 5-iodo-20-
deoxyuridine. ONs modified with monomer X allow efficient
fluorescent discrimination of SNPs via a G-specific quenching
mechanism, while ONs modified with monomer Y display
remarkable hybridization-induced increases in emission and
discrimination of Y:G-mismatches. Evaluation of these building
blocks in diagnostic assays is ongoing.
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We appreciate financial support from Idaho NSF EPSCoR,
the BANTech Center at Univ. of Idaho, a University of Idaho
Research Office and Research Council Seed Grant, and a
scholarship from the College of Graduate Studies, Univ.
of Idaho (M.E.Ø). We thank the EBI Murdock Mass
18 S. Tyagi, Nat. Methods, 2009, 6, 331–338.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 4929–4931 | 4931