A rapidly photo-activatable light-up fluorescent nucleoside and its application in DNA base variation sensing

Article abstract of DOI:10.1039/c6cc03098j

A new DNA building block (d^TetU) bearing a tetrazole and allyloxy group at N-phenyl ring linked through an aminopropynyl linker to the 5-position of 2′-deoxyuridine was synthesized. The modified DNA can be lit up via a photoinduced intramolecul

Full text of DOI:10.1039/c6cc03098j

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DOI: 10.1039/C6CC03098J
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A Rapidly Photo-activatable Light-up Fluorescent Nucleoside and
Its Application in DNA Base Variation Sensing
Received 00th January 20xx,
Accepted 00th January 20xx
Zhiyong He, ‡ Yuqi Chen, ‡, Yafen Wang, Jiaqi Wang, Jing Mo, Boshi Fu, Zijing Wang, Yuhao Du*
and Xiang Zhou*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Tet
A new DNA building block (d U) bearing a tetrazole and allyloxy nucleotide polymorphisms (SNPs) due to their sensitivity to
group at N-phenyl ring linked through an aminopropynyl linker to
the 5-position of 2’-deoxyuridine was synthesized. The modified
DNA can be lighted up via photoinduced intramolecular tetrazole-
alkene cycloaddition reaction, but quenched when formed fully-
matched double strand. This conspicuous difference in
fluorescence could open a door for DNA single nucleotide
polymorphisms (SNPs) typing.
Fluorescence labelling of nucleic acid especially on the base
has become increasingly important in biophysical studies of its
structure, function, and dynamics. Post-synthetic modification
is one of the most common methods for labelling nucleic acids.
There has been considerable interest in the development of
nucleic acid building blocks, each bearing a specific reactive
functional group that allows the attachment of various
different fluorophores after nucleic acid synthesis. Copper(I)-
catalyzed cycloaddition of azides and alkynes (CuAAC) has
Tet
Scheme 1 Photo-induced intramolecular cycloaddition of d U.
5
found favour in labelling oligonucleotides with fluorescence oligonucleotide hybridization and conformation change. Since
over the last decade. Due to the high cytotoxicity of copper SNPs in a protein- encoding region may cause protein
1
6
ions, it is limited to application in vivo. Hence, copper free mutation and even result in diseases, the typing of SNPs using
7
strain-promoted azide–alkyne cycloaddition (SPAAC) was base-discriminating fluorescent nucleosides (BDFs) and
established in the field of nucleic acid research, building blocks fluorescent phosphate backbone became an active area of
containing dibenzocyclooctyne or other cyclooctyne analogues research. Most of these fluorescent nucleobases can be
8
9
at position 5 and at position 2’ of uridines were found to label broadly classified into two categories : (a) conjugation of
2
DNA with fluorescent azide probe in a few minutes.
fluorescent molecules such as pyrene or fluorene to
Afterwards, a more selective and efficient method for deazapurine and pyrimidine bases and (b) natural nucleobases
modification of oligonucleotides has been explored by using that are extended by conjugation to additional aromatic rings.
the inverse electron demand Diels–Alder reaction of tetrazines
and alkenes.
tetrazine or norbornene group was photoinduced cycloaddition of biaryl-substituted with alkenes
incorporated into DNA by solid-phase synthesis then3 as an interesting bioorthogonal photoclick reaction . Later,
Recently, Lin and co-workers described
a
rapid
A
1
0
11
conjugated with corresponding fluorophore labelling reagent.
the reaction was adapted to fluorescently labelling proteins
1
2
Another method for fluorescence labelling nucleic acids is and post-synthetic modification of nucleic acids. Due to the
designing fluorescent nucleosides or fluorophore-linked base excellent biocompatibility and rapid reaction kinetics of the
conjugates then incorporating them into oligonucleotides reaction, it could provide us a valuable labelling strategy for
4
through solid phase synthesis or polymerase synthesis. Such DNA nucleobases extending.
fluorescent nucleoside analogues are widely used in single
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Tet
Fig. 1 Time courses of of photoactivation of d U containing
oligodeoxynucleotide ODN(1) (a) Fluorescence enhancement as the
irradiation time going (b) Fluorescence emission at 530 nm plotted
against irradiation time and product concentrations plotted against
irradiation time according to the rate law (pseudo-first order), a
Tet
Scheme 2. Synthesis of d U phosphoramidite 11. a) NHS, EDC,
DCM 0°C to RT,10h, 95%; b) 2,2,2-trifluoro-N-(prop-2-yn-1-yl)
acetamide, CuI, Pd(PPh
3
)
4
, Et
M ammonia solution in methanol, quant.; d) DIPEA, DMAP, DCM,
h, 93%; e) N,N,N',N'-tetra-isopropylphosphorodiamidite,
NHS = N-hydoxy-
3
N, anhydrous DMF, 50°C, 12h, 81%; c)
1
4
-1
2
linear fit to get a value for rate constant kobs= 0.0483s , R =0.9945.
c) HPLC spectra at different time monitoring the reaction. ODN(1)
(
tetrazole, anhydrous DCM, Ar, 0°C, 45%.
was dissolved in 10mM PBS (pH7.4) to derive concentration of 10
μM. Photoirradiation wavelength was set at 305 nm for ODN(1) .
For fluorescence, λex = 330 nm.
succinimide EDC= 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
DCM=dichloromethane, DMF= N, N -Dimethylformamide DIPEA=
diisopropylethylamine, DMAP= 4-dimethylaminopyridine, DMTr =
dimethoxytrityl.
Herein, we envisioned a conceptually new method for procedures. ODN(1) was synthesised from this DNA building
nucleic acid fluorescence labelling with a fluorescence turn-on block by automated solid-phase chemistry and purified by
Tet
assay. We described the new DNA building block d U, which semi-preparative HPLC.
bears the biaryl-tetrazole and ortho-allyloxy group at N-phenyl Subsequently, the fluorescence spectra was employed to
ring linked through an aminopropynyl group to the 5-position study the irradiation kinetics for photoclick chemistry of the
of 2’-deoxyuridine, exhibiting a strong fluorescence emission OND(1). First, single strand ODN(1) in standard aqueous buffer
after UV irradiation subsequent forming d U (Scheme 1). solution (10 mM PBS buffer, pH 7.4) was exposed under 305
Prl
When there is a base variation at the complementary strand nm hand-held lamp, after irradiation, fluorescence was turned
opposite site of d U, it showed significantly different on within 5s according to the fluorescence spectra(Fig. 1a).
Prl
fluorescence signal and intensity. Thus, oligonucleotide With excitation of the single strand DNA at 330 nm, we
containing d U can be used for the detection of single base observed strong fluorescence at 530 nm. However, its
Tet
variation, together with its incorporation into an fluorescence increases approximately 21-fold in only about
oligonucleotide by using automated phosphoramidite 1min. An absorption band at 364nm appeared indicating the
chemistry.
formation of pyrazoline fluorophore (Figure S4). Moreover,
U phosphoramidite 11 was prepared following the this intramolecular reaction was monitored by analytical HPLC
protocol depicted in Scheme 2. 5’-dimethoxytrityl-5-iodine 2’- technology, the chromatograms of which are displayed in Fig
deoxy-uridine ( ) was adopted for consideration of easiness 1c. After 10 s of reaction, an obvious new peak (rt= 17 min)
Tet
d
7
for nucleoside derivative compounds separation at silica gel was observed, and with the increase of time, this new peak
column. The synthesis commenced with Sonogashira cross- increased in intensity as ODN(1) peak (rt=18.5 min)
coupling reaction of 7 and 2,2,2-trifluoro-N-(prop-2-yn-1- disappeared within 60 seconds. In addition, the product was
yl)acetamide, then the trifluoroacetyl group was removed by identified by MALDI-TOF mass spectrometry (Figure S3). The
ammonia solution in methanol, thus 5’-dimethoxytrityl-5-(3’’- MS analysis results showed that during this process one
aminopropynyl)-2’-deoxy-uridine(
precursor. The other precursor, TetI- succinimide activated
ester was synthesised from TetI and N- well on double strand DNA, the fluorescence spectra of
9
) to be obtained as one molecular of nitrogen released.
To investigate whether this intramolecular reaction works
(
6)
(5)
hydroxysuccinimide. The activated ester moiety of
attached to the aminopropynyl group of nucleoside
6
9
was corresponding oligodeoxynucleotide duplexes ODN(1)/ODN(A)
with were obtained after irradiation Figure S5). Obvious
(
DIPEA. Then converted into DNA building block 11 by standard fluorescence quenching was observed in spite of longer time
2
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light for 1min (Table S1). It might result from structure rigidity.
Prl
Tet
The structure of d U is more rigid than d U. The difference in
fluorescence intensity may be explained with this as well. The
higher T
m
value indicated the stronger hydrogen bonds
interaction between the base pair, thus the fluorescent
intensity should be lower due to restriction of pyrazoline
moiety in the groove and the stack effect with the adjacent
[13]
[8]
base .
To further study the potential application of this
modification of DNA probe in the detection of single AP site.
Sensitivity and accuracy were further verified by detecting
mixed samples with diverse AP site sample levels. Samples of
0
%, 5%, 10%, 50%, 100% AP site containing DNA were diluted
Fig. 2 Fluorescent spectra of oligodeoxynucleotides containing
Tet
with fully matched DNA for a total of 10μM of input DNA. All
samples were subjected to 302 nm irradiation for 1 min. The
fluorescence intensity respectively increased in a relatively
linear relationship with the proportions of AP sample. It also
showed evident and gradual colour change visually under 365
nm illumination.
d
U and and the corresponding duplexes after 302 nm
330 nm. Concentration of each oligo-
deoxynucleotide was 10 μM. All irradiation experiments were
irradiation,
ex
λ =
o
taken at 25 C for a total irradiation time of 1 min, in 10 mM PBS
buffer, pH 7.4.
In summary, we have devised a simple dU analogue, which
could achieve fluorescence turn-on under UV irradiation within
just 1 min. This strategy could be a new way to label nucleic
acid with fluorescence rapidly without any other fluorescent
reagents adding. It potentially allows this nucleoside conjugate
with biaryl-tetrazole and ortho-allyloxy to be used in the
[13]
Prl
enzymatic preparation of oligonucleotides . The d U display
different emission in duplex versus single-stranded
a
Prl
oligonucleotides. Furthermore, d U responded sensitively
when single nucleotide alteration and abasic sites occur in
complementary strand. The particular significance is its ability
Fig. 3 (a) Fluorescence spectra of duplexes with different fraction
of AP site. Concentration of ODN(A) changes from 0 to 10 μM,
to report the presence of adenine sites with remarkably
quenched fluorescence intensity. This method using d
Tet
U
while concentration of ODN(AP) from 10 μM to 0, , λ =330 nm.
ex
containing probes is a very flexible homogeneous assay that
does not require enzymes or time-consuming steps, and is
potential for single base mutation detection.
(b) Linear relationship between intensity of fluorescence
emission and the fraction of AP sample, R =0.9937. All
irradiation experiments were taken at 25 C for a total irradiation
time of 1 min, in 10mM PBS buffer, pH 7.4.
2
o
Acknowledgements
irradiation compared to single strand DNA. Besides the
emission bathochromic shift from 525nm to 538nm, double
strand sample became a pale pink solution under 365m
illumination.
Furthermore, we estimated the influence of d U on both
complementary and non-complementary (mismatched)
This work was supported by the National Basic Research
Program of China (973 Program) (2012CB720600,
2
012CB720603), the National Science Foundation of China
nos 21432008, 91413109, 81373256, 21402147) and the
National Grand Program on Key Infectious Disease
Prl
(
duplexes. In the duplexes of ODN(1) with mismatched strands, (2012ZX10003002-014). We also thank Prof. Zhenjun Yang at
the fluorescence spectra of d U was strongly dependent on Peking University for the guidance with the ongoing project.
Prl
the base paired with it. ODN(1)/ODN(G), ODN(1)/ODN(T) and
ODN(1)/ODN(C) duplex didn’t show fluorescence until they
Notes and references
Prl
were irradiated and d U formed. The fluorescence intensity of
mismatched ODN(1)/ODN(G) was inhanced 2-fold vs that of
ODN(1)/ODN(T)
1
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Tet
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