A diazirine-based photoaffinity probe for facile and efficient aptamer-protein covalent conjugation

Article abstract of DOI:10.1039/c4cc01528b

A photo-reactive functional labelling reagent, diazirine phosphoramidite, was designed and synthesized for easy and flexible site-specific labelling of oligonucleotides with the diazirine moiety. The new reagent allows facile photo-crosslinking of oligonu

Full text of DOI:10.1039/c4cc01528b

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A diazirine-based photoaffinity probe for facile
and efficient aptamer–protein covalent
conjugation†
Cite this: Chem. Commun., 2014,
50, 4891
Received 28th February 2014,
Accepted 21st March 2014
Huimin Zhang, Yanling Song, Yuan Zou, Yun Ge, Yuan An, Yanli Ma, Zhi Zhu* and
Chaoyong James Yang*
DOI: 10.1039/c4cc01528b
www.rsc.org/chemcomm
A photo-reactive functional labelling reagent, diazirine phosphor- aptamer and the target. If this non-covalent interaction can
amidite, was designed and synthesized for easy and flexible site- be transformed into a covalent bond, the stable aptamer–target
specific labelling of oligonucleotides with the diazirine moiety. The complex can be obtained with higher purity by harsh washing,
new reagent allows facile photo-crosslinking of oligonucleotide greatly facilitating the process of biomarker discovery on
with its interacting partner for a variety of applications, including cell membranes.
tertiary structure determination, molecular interaction study and
biomarker discovery.
Photoaffinity probes are promising ligands for the formation
of the covalent bonding with good spatial and temporal resolution.
Currently, they have been widely used to covalently link protein
The identification of biomarkers associated with disease states with small molecule or another protein to study their interactions.5
is extremely important for disease diagnosis and treatment. The probes contain at least two parts: an affinity unit and a photo-
The combination of chromatographic techniques with mass reactive moiety.6 Azide, benzophenone and diazirine are common
spectrometry (MS), which enables researchers to analyse the photo-reactive groups that can generate highly reactive species
entire cell proteome and identify cell-specific proteins, has such as nitrene, reactive triplet carbonyl state and carbene under
become one of the most widely used methods for biomarker UV irradiation.7 Among them, azide is most widely used because
discovery.1 Although this method has enabled the discovery of it is easy to synthesize.8 However, it generates nitrene that could
several important proteins, it is time-consuming, labor-intensive lead to the undesired side product of ketimines.9 Moreover,
and inefficient. Thus it is highly desirable to develop rapid, azide requires the irradiation with destructive short-wavelength
facile, and efficient methods for biomarker discovery.
UV (o300 nm). Benzophenone can be photo-activated by long-
Recently, an aptamer-based ‘‘fishing’’ method was developed for wavelength UV (B350 nm) but needs a long photo-irradiation
identification of diseased cell related biomarkers.2 Aptamers are time for the reaction.10
single-stranded oligonucleotides generated by a well-established
Diazirine is one of the most effective photo-reactive groups
method named SELEX (Systematic Evolution of Ligands by because it can generate a highly active carbene intermediate
EXponential enrichment).3 When using whole disease cell as which can even be inserted into the inactive C–H bond.7,11
the target, a panel of aptamers can be generated by cell-SELEX Meanwhile, the photoreaction of diazirine uses long-wavelength
without the knowledge of cell membrane characteristics.4 Then, UV and short irradiation time, which causes less damage to
specifically bound molecules on the cell surface can potentially biomolecules and minimizes non-specific labelling. Therefore,
be identified as biomarkers using the aptamer-based ‘‘fishing’’ diazirine has been widely used for a variety of photo-labelling,
method. Although it is a straightforward and promising method such as the covalent linking of a ligand to its target for the
for biomarker discovery, one major challenge is the dissociation investigation of ligand–receptor interactions,12 identification of
of the aptamer–target complex during the identification process enzyme inhibitor binding sites,13 and discovery of the key amino
due to the unstable non-covalent interactions between the acid residues in protein–protein interactions.14 Various diazirine
analogs of nucleic acids have also been developed for DNA
The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation,
Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of
Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of
Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005,
P. R. China. E-mail: cyyang@xmu.edu.cn, zhuzhi@xmu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc01528b
modification, such as 2,3-dideoxyuridylate with a diazirine on
the C5-position.15 A simpler and more popular method for DNA
labelling is based on the coupling of an amine-modified oligo-
nucleotide with the succinimide ester of diazirine. Unfortunately,
the low reaction efficiency and modification site restriction greatly
impede further application of these modified oligonucleotides.
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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
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Fig. 3 SDS-PAGE analysis of photoaffinity labelling of SA with diazirine-
labelled-aptamer in the presence of a high background protein (BSA).
M: marker.
the hairpin-loop structure of the SA–aptamer. To find the most
suitable photo-crosslinking site, we inserted diazirine into four
different sites, and used the same method to test whether the
probe could covalently link to the target protein. As shown in
Fig. 2D, although there was a slight decrease in the binding
capacity of TBA-25-drz, it can still be covalently linked to target
after UV irradiation. And the other modified aptamers cannot
form covalent binding with the protein beads due to the loss of
binding affinity (Fig. S3, ESI†).
Fig. 2 (A) Binding of aptamer sequences SA and SA-6-drz to SA beads
before and after UV/urea treatment. (B) Effect of UV irradiation time on the
crosslinking efficiency of modified aptamer sequence SA-6-drz to SA
beads. (C) Comparison of the cross-linking efficiency between diazirine
and I-dU modified probes under their respective optimized conditions. (D)
Binding of aptamer sequences TBA and TBA-25-drz to thrombin beads
before and after UV/urea treatment.
In conclusion, we have designed and synthesized a photo-
crosslinking reagent, diazidite, which can be used for site-
specific labelling of aptamer sequences directly by the general
DNA synthesis protocol. The synthesis of diazidite is simple and
straightforward. Diazirine has the advantages of high crosslinking
efficiency, small size and excellent chemically stability, which are
highly desirable for use in aptamer labelling. We demonstrated
that by choosing the right modification position, the diazirine
modified streptavidin and thrombin aptamers have high efficiency
and specificity for photo-crosslinking with their corresponding
target proteins. The diazidite probe is thus useful for biomarker
discovery by providing a novel and efficient strategy for covalent
labelling of biomarkers with aptamers generated from cell-SELEX.
Moreover, because it can be inserted into any site of a DNA/RNA
sequence, the diazidite is potentially useful for the determination
of DNA/RNA secondary structures and identification of nucleic
acid–protein interaction binding sites.
efficiency of the diazidite was then compared with the com-
monly used I-dU modification. To ensure fair comparison, the
probe SA-6-I-dU was synthesized using the same process as that
of SA-6-drz. SA-beads were incubated with 200 nM of either
SA-6-I-dU or SA-6-drz for 30 min at 37 1C, and then irradiated
with 308 or 365 nm UV light at the same power on ice,
respectively. As shown in Fig. 2C, with all three irradiation
time durations (2, 5 and 10 min), the SA-6-drz treated sample
displayed a significantly higher crosslinking efficiency than that
of SA-6-I-dU, demonstrating the excellent labelling efficiency of
the diazirine over the I-dU based photoaffinity probe.
In order to validate whether SA-6-drz can photo-crosslink
with SA in the high background of irrelevant proteins, the reaction
of SA-6-drz and SA was performed in the presence of ten-fold
concentrations of bovine serum albumin (BSA), and the result was
analysed by SDS-PAGE. Because noncovalent interactions are dis-
rupted in SDS-PAGE, only covalent bonding as the result of photo-
crosslinking will result in the formation of the protein–aptamer
complex. The SDS-PAGE results were obtained by recording the
fluorescence image of FITC-labelled SA-6-drz. As shown in Fig. 3,
after UV irradiation, a band corresponding to streptavidin–aptamer
complexes (25 kD) was observed (lanes 4–6) and it increased
with irradiation time, but there was no BSA–DNA complex band
(B75 kD) even with 1-, 5- or 10-fold excess BSA (lanes 7–9).
These results revealed that diazirine modified aptamer had
high selectivity for photo-crosslinking with the target protein,
suggesting the potential of the diazidite for use in biomarker
discovery with aptamers generated from cell-SELEX.
We thank the National Basic Research Program of China
(2010CB732402, 2013CB933703), the National Science Foundation
of China (91313302, 21205100, 21275122, 21075104), the Funda-
mental Research Funds for the Central Universities 2012121025,
and the National Science Foundation for Distinguished Young
Scholars of China (21325522) for their financial support.
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