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With 4-40-dimethoxytriphenylmethyl chloride (DMT-Cl), one of
the hydroxyl groups was then protected by DMT, while the other
hydroxyl was coupled with 2-cyanoethyl diisopropylchloro-
phosphoramidite to generate the final product (5). The DMT-
protected diazidite is highly compatible with the general DNA
synthesis protocol. Moreover, ten carbons have been introduced
between the diazirine group and the backbone of DNA to enable
appropriate flexibility for diazirine to react with targets.
The as-synthesized diazidite was applied to label the strep-
tavidin aptamer17 as an example to demonstrate the feasibility
for covalent binding with streptavidin (SA) by UV irradiation.
Since the highly active carbene intermediate generated by
diazirine can react only with nearby molecules, the suitable
diazirine labelling sites on the aptamer needed to be identified
for highly efficient photo-crosslinking with the target, while
maintaining the binding affinity. According to the secondary
structure of the SA–aptamer, three sites were chosen. The resulting
diazirine modified SA–aptamers (Table S1, ESI†) were synthesized
and labelled with FITC for easy characterization. SA-beads were
used as targets so that the formation of covalent bond could be
directly detected by flow cytometry. The modified SA–aptamers
were incubated with SA-beads in the dark at 37 1C for 30 min. After
washing away the excess or nonspecific sequences, the resulting
complexes were irradiated with 365 nm UV light on ice. Afterwards,
the complex was subjected to 10 M urea washing. Since urea can
destroy the non-covalent interaction of the SA–aptamer with SA,
the non-crosslinked SA–aptamers could be washed away from the
SA-beads, leaving the beads nonfluorescent, while the covalently
bound aptamers stayed with SA, yielding fluorescent beads. Flow
cytometry was used to detect the fluorescence intensity of SA-beads
after different treatments.
First, the effect of the modification site on aptamer binding
affinity was investigated. As shown in Fig. S1 (ESI†), compared
with the unmodified SA–aptamer, different insertion locations
of diazirine have distinct influence on aptamer binding. This
suggested that the inserted diazidite may disturb the secondary
structure of aptamer and affect its binding affinity. Then, the
crosslinking capability of each modified aptamer was evaluated
(Fig. S2, ESI†). Among them, SA-6-drz has shown the best
crosslinking efficiency. As shown in Fig. 2A, after treatment with
10 M urea, the fluorescence intensity of SA-beads with unmodified
SA–aptamer was greatly decreased due to the destruction of
noncovalent interactions by urea. However, the fluorescent
intensity of SA-beads with SA-6-drz did not exhibit a significant
change after UV irradiation and washing with 10 M urea, suggesting
that the covalent bond was successfully formed between the aptamer
and the protein. To verify that the covalent bond formation was
indeed the result of UV irradiation, the SA-6-drz sample was treated
with different irradiation times. In Fig. 2B, as the irradiation time
increased, the fluorescence intensity of SA-beads treated with urea
also increased, indicating that the quantity of covalent complexes
increased with longer irradiation. These results demonstrated that
diazirine labelled aptamer could still bind the protein and formed
the covalent bond by photo-irradiation.
Scheme 1 Photo-initiated efficient covalent coupling of diazirine modified
aptamer probe with its target protein for biomarker discovery.
Therefore, it is of great importance to develop diazirine probes
for simple, facile and efficient labelling on DNA ligands, such
as aptamers, for DNA–protein interaction study and aptamer-
based biomarker discovery.
Herein we developed a new diazirine probe for simple, facile
and efficient labelling of DNA ligands. Our diazirine phosphor-
amidite (diazidite) can be chemically synthesized by a simple
procedure and used for easy and flexible site-specific labelling
of a DNA sequence with an automated DNA synthesizer. Using
the resulting diazirine-labelled aptamer to interact with the
target, a covalent bond can be formed between the aptamer and
the target by 365 nm irradiation. By fishing out such covalent
complexes and analysing them with the help of MS, the possible
biomarkers on the disease cell surface can then be discovered.
According to the principle described in Scheme 1, diazirine is
inserted into a certain position of the aptamer, and the high
affinity and selectivity of the aptamer enable specific capture of
target protein. As a proof-of-concept, we adopted two known
aptamer targets, streptavidin (SA) and thrombin (TMB) to verify
the feasibility of photo-crosslinking capability of diazirine-labelled
aptamers with target proteins. Additionally, we compared the
photo-crosslinking efficiency of our probe with that of the widely
used I-dU probe.
The synthesis of diazidite is shown in Fig. 1. The diazirine–
COOH (2) was synthesized from 4-oxopentanoic acid (1) by
reacting with NH2OSO3H in liquid ammonia and oxidized by
iodine.16 The diazirine–COOH was reacted with 6-amino-2-
hydroxymethylhexan-1-ol to obtain two hydroxyl groups.
Fig. 1 Synthesis of diazirine phosphoramidite: (a) 1 NH3 (liquid), NH2O-
SO3H, MeOH; 2 I2, Et3N, MeOH, 0 1C; (b) DCC, HOBt, DMF, room
temperature, 24 h; (c) DMT-Cl, pyridine, CH2Cl2, DMAP, ice bath, and
then room temperature about 24 h; (d) P-(N-iPr2)2(OCH2CH2CN), DIPEA,
CH2Cl2, 0 1C, about 4 h.
Previously, I-dU was used for photoaffinity labelling of
aptamers for biomarker discovery.18 The photo-crosslinking
4892 | Chem. Commun., 2014, 50, 4891--4894
This journal is ©The Royal Society of Chemistry 2014