Introduction
Vitamin D plays a vital role in bone health through regulation of
calcium and phosphorus homeostasis, which can facilitate intestinal
calcium absorption and provide calcium necessary for bone
mineralization.[1,2] Prolonged and severe
deficiency of vitamin D leads to rickets (in children) and osteomalacia
(in adults).[3,4] Besides, epidemiological studies
have increasingly found that vitamin D deficiency link with a wide range
of non-communicable diseases such as obesity, hypertension, diabetes,
heart failure.[5-7] Vitamin D is synthesized when
exposed to ultraviolet radiation (UVR) through the skin epidermis
(vitamin D3), or absorbed from food or supplements
(vitamin D2 and vitamin
D3).[8] The vitamin
D3 is hydroxylated by the liver enzyme 25-hydroxylase to
25-HydroxyvitaminD3 [25(OH)D3,
cholecalciferol], half-activated form of vitamin D3.
When the certain tissue in our body needs vitamin D function,
25(OH)D3 is converted by 1-α-hydroxylase to
1,25-dihydroxyvitamin D3[1,25(OH)2D3, calcitriol], which is
a fully activated form.[9,10] As the major
circulating form of vitamin D, 25-HydroxyvitaminD3 is
considered to be the indicator of vitamin D
status.[11] The serum vitamin D3levels can be assessed through measuring the concentration of
25-HydroxyvitaminD3.[12]
The standard quantitative detection methods for vitamin
D3 levels mainly include high-performance liquid
chromatography (HPLC) and enzyme-linked immunosorbent assay
(ELISA).[13,14] HPLC method requires tedious
pretreatments, sophisticated equipments and trained personnel, giving
rise to increasing analysis time. Although with good sensitivity and
selectivity, ELISA may suffer from matrix interference, false positive
results and large deviations in qualitative results. Besides, some
commercial antibody kits are applied for
25-HydroxyvitaminD3 detection, however with the antibody
limitations of high cost, complicated storage condition and short shelf
life.[12] In comparison, aptamers (Apt), highly
specific oligonucleotides, can bind to a variety range of target ligands
and are used as an alternative to
antibodies.[15-18]
Aptamers are single-stranded DNA (ssDNA) or RNA molecules which are
selected in vitro by systematic evolution of ligands via an exponential
enrichment (SELEX). They can bind to various target ligands ranging from
small molecules, peptides, proteins to cells with high affinity and
specificity.[19] Compared with traditional
antibodies, aptamers possess notable advantages including high binding
capability, excellent stability, target diversity, low cost, simple
synthesis and easy modification.[20,21]Aptamer-based detection system are non-immunogenic, low toxicity and
stable to temperature and pH changes.[22] However,
many selected original aptamers with long sequences possess poor
affinity and are limited in application. Thus, truncation of aptamers is
valuable work, for that many aptamers lying in the corner can be used
with high performance after truncation.
Lee et al., have developed novel aptamers for detecting
25-HydroxyvitaminD3by graphene oxide-based systemic evolution of ligands by exponential
enrichment (GO-SELEX) and found the aptamer VDBA14
(5’-AGCAGCACAGAGGTCATGGGGG-GTGTGACTTTGGTGTGCCTATGCGTGCTACGGAA-3’, 56
bases) showing specific affinity to 25-HydroxyvitaminD3,
with the limit of detection of 1 μM.[12] In order
to increase the aptamer affinity to its target and avoid large steric
hindrance of long aptamer sequence, a few simple or heuristic approaches
towards the truncation of aptamers were reported, other than addition to
optimize the length of the random library used in the SELEX
process.[23] In this study, we have heuristically
truncated the VDBA14 to four aptamers with different sequences by
intercepting the small hairpin loop and retaining double helix regions
of different lengths that exist in VDBA14 aptamer, while maintaining
higher affinity to 25-HydroxyvitaminD3. A universal
PicoGreen-based fluorescence strategy for the quantitative detection of
25-HydroxyvitaminD3 was designed, that the fluorescent
signal of PG generated upon the double-stranded DNA (dsDNA) formed
between the aptamer and its complementary strand. Coupled with the
simple PG-based fluorescent assay, the analytical results indicated that
the truncated aptamers showed higher affinity than the original aptamer,
and exhibited great potential for sensitive detection of
25-HydroxyvitaminD3.
Materials and methods
Materials and reagents
All the oligonucleotide sequences used in this study were synthesized
and then purified through HPLC by Shanghai Sangon Biotechnology Co., Ltd
(Shanghai, China), and sequence information were listed in Table 1. The
lyophilized powder of aptamers and complementary strands were dissolved
in ultrapure water to 10μM and then diluted to 0.5μM in binding buffer
(100 mM NaCl, 20 mM Tris-HCl, 2 mM MgCl2, 5 mM KCl, 1 mM
CaCl2, pH 7.6). The PicoGreen (PG) dsDNA reagent was
purchased from Thermo Fisher Scientific (Shanghai, China).
25-HydroxyvitaminD3 monohydrate was obtained from
Sigma-Aldrich International GmbH. Vitamin C and Vitamin B12 were
purchased from TMstandard (Beijing, China). Folic acid was obtained from
Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). All other
reagents were of analytical grade, and the
ultrapure water used throughout
the experiments was purified by Milli-Q Academic system (Millipore,
Molsheim, France). The fluorescence intensities were recorded with a
Tecan Infinite 200 multifunctional microplate reader (Tecan Austria
GmbH, Austria), with the excitation and emission wavelengths of 480 nm
and 520 nm, respectively.
Truncation for 25-HydroxyvitaminD3 aptamer
The original aptamer with long oligonucleotide sequences may bind
analogues of its targets through primer region or additional fragments,
resulting in compromised selectivity.[24] It is
significant to confirm the target binding region where the aptamer
undergoing conformation change upon target binding. Thus, truncation of
the non-binding region for original aptamer may generate aptamers with
better performances in consideration of affinity, selectivity and cost.
Several studies reported on aptamer engineering approaches were focused
on the G-quadruplex region, secondary structure (stem-loop) and
stem-loop multiplication, on account of stem-loop known to be the
binding motif for most proteins.[23] Based on
molecular shape and functional base distribution of
25-HydroxyvitaminD3, the loop regions of original
aptamer were assumed to be the functional domain for binding to
25-HydroxyvitaminD3target. In this study, the original VDBA14 aptamer was truncated on the
basis of retaining small hairpin loops. Then the experiments for
affinity and specificity were performed to ascertain the functional and
structural domains of original aptamer.
Principle of the fluorescent assay
PicoGreen (PG) reagent is an asymmetric cyanine dye that does not
fluoresce when free. However, the fluorescence of this commercially
available dye increases by more than 1000-fold when upon binding to
dsDNA, while no significant fluorescence change can be observed after
binding to ssDNA.[25] Here, we performed a
universal PG-based fluorescence strategy for the quantitative detection
of 25-HydroxyvitaminD3. The sensing mechanism of the
method was illustrated in Figure 1. In the control group, dsDNA was
formed through hybridization of
25-HydroxyvitaminD3-specific aptamer and corresponding
complementary strand, then PG dye inserted into the minor groove of
dsDNA duplex, that resulting in significant enhancement of
fluorescence.[26,27] In the test group, specific
binding of 25-HydroxyvitaminD3 and aptamers occurred
prior to dsDNA formation, and the small amount of remaining free
aptamers reacted with complementary strands, which generating a decrease
in fluorescence intensity compared to the control group. Thus,
quantitative analysis of 25-HydroxyvitaminD3 can be
accomplished by monitoring the fluorescence intensity changes of PG dye.
Screening of aptamers for 25-HydroxyvitaminD3
The screening of four truncated aptamers (D3-1, D3-2, D3-3 and D3-4) and
the original aptamer (VDBA14) for 25-HydroxyvitaminD3were carried out in this work, based on the analysis foundations of
affinity and specificity. Although dissociation constant (KD) can be
applied to evaluate affinity between aptamers and target ligands, it
fails to describe specificity and cross‐reactivity. Therefore, we
performed the aptamers screening for 25-HydroxyvitaminD3with high affinity and specificity by means of a simple fluorescent
strategy using commercial PG dye. Through comparison for the affinity
and specificity of the four truncated aptamers and the original aptamer,
suitable aptamer with highest affinity and best specificity to
25-HydroxyvitaminD3 was identified.
Fluorescent detection of 25-HydroxyvitaminD3
To determine the sensitivity of this method, different concentrations of
25-HydroxyvitaminD3 standard solution were progressively
diluted from 10μg/mL in binding buffer (100 mM NaCl, 20 mM Tris-HCl, 2
mM MgCl2, 5 mM KCl, 1 mM CaCl2, pH 7.6).
10μg/mL 25-HydroxyvitaminD3 was obtained through
dissolving the lyophilized powder of 25-HydroxyvitaminD3monohydrate in methanol. 25μL of 0.5μM aptamer solution and 50μL of
different concentrations of 25-HydroxyvitaminD3 were
successively added into a microplate well and incubated at room
temperature for 40 min. Afterwards, 25μL of 0.5μM complementary strand
solution was added to the microplate well and incubated for 10min. Then
10μL 10×PG solution was added, and after 3min incubation, the
fluorescent intensities were recorded with a multifunctional microplate
reader. Each concentration was performed in triplicate to obtain average
values for analysis.
Results and discussion
Aptamer truncations and sequence designs
With the aim to ascertain the binding sites of original VDBA14 aptamer
and to obtain the truncated sequences with increased affinity, the
secondary structure analysis of original aptamer is essential. In this
study, two secondary structures of the original aptamer for
25-HydroxyvitaminD3(VDBA14) were obtained from mfold software program (shown in Figure 2A
and Figure 2B).
Based on the first secondary structure of original VDBA14 aptamer, we
designed two truncated aptamers (denoted as D3-1 and D3-2) by rationally
intercepting the sequences containing small hairpin loop and retaining
double helix structural domains of different lengths. In the double
helix region of D3-1 and D3-2, the G-T mismatched pairs were replaced by
G-C base pairs, and one A-T base pair was replaced by G-C base pair in
D3-2 aptamer to strengthen the stability of the whole structure
(displayed in Figure 2C and 2D). There is an alternative small loop in
another secondary structure of original VDBA14 aptamer (Figure 2B), and
then D3-3 and D3-4 (Figure 2E and Figure 2F) were obtained by
substitution of this small loop with loop regions in D3-1 and D3-2. The
secondary structures of all aptamers predicted by the mfold program were
shown in Figure 2, and the sequences for
25-HydroxyvitaminD3 aptamers and corresponding
complementary strands used in this study were listed in Table 1.
Optimization of PG concentration for different aptamers
The fluorescent assay of 25-HydroxyvitaminD3 was based
on a combination of PG dye and
aptamer/complementary
strand-formed dsDNA. Thus, the concentration of PG fluorescent dye is an
important parameter influencing the detection performance. The PicoGreen
dsDNA reagent was diluted 5-folds, 10-folds, 20-folds, 30-folds and
40-folds by 1×TE buffer, that diluted PG reagents were labeled as 5×,
10×, 20×, 30× and 40×. After adding 25μL of 0.5μM aptamer solution and
50μL binding buffer to a microplate well, 25μL of 0.5μM complementary
strand was added and incubated at room temperature for 10min. Then 10µL
of 5×, 10×, 20×, 30× and 40×PG solution were added into the mixture at
the last reaction section. As displayed in Figure 3, the fluorescence
intensities gradually increased with the increase of PG concentration.
For VDBA 14, D3-3 and D3-4 aptamers, the fluorescence intensities
reached basically stable peak and did not change obviously when 10×PG
were added, indicating that 10× PG were enough to mark dsDNA after
binding of aptamers and complementary strands. For D3-1 and D3-2
aptamers, increasing the PG concentration did not lead to an obvious
intensity increase when adding 20×PG. Thus, the selected PG
concentrations for VDBA 14, D3-1, D3-2, D3-3 and D3-4 were 10×PG, 20×PG,
20×PG, 10×PG and 10×PG, respectively.
Screening of 25-HydroxyvitaminD3‐specific
aptamers
In this study, we compared the affinity of original aptamer and four
truncated aptamers to 25-HydroxyvitaminD3 through
PG-based fluorescent assay, and selected the aptamers with high affinity
for further detection of 25-HydroxyvitaminD3. The
comparison results for aptamer affinity were shown in Figure 4A, in
which ΔF stand for the decreased value of fluorescence intensity when
aptamer specifically bound to 25-HydroxyvitaminD3, and
F0 stand for the fluorescence intensity in the absence
of 25-HydroxyvitaminD3. Demonstrated in Figure 4A,
compared with VDBA14 aptamer, four truncated aptamers of D3-1, D3-2,
D3-3 and D3-4 gave rise to significant decrease of fluorescence
intensity, indicating these four truncated aptamers possessed higher
affinity to 25-HydroxyvitaminD3 than original VDBA14
aptamer. In particular, D3-3 and D3-4 had strongest affinity to
25-HydroxyvitaminD3 with distinct fluorescence intensity
decrease than D3-1 and D3-2. Therefore, D3-3 and D3-4 aptamers were used
for following specificity evaluation.
Specificity of the
selected aptamers for 25-HydroxyvitaminD3
To evaluate the specificity of D3-3 and D3-4 aptamers, the fluorescence
intensity changes were induced against
25-HydroxyvitaminD3 and its analogues including vitamin
C, vitamin B12 and folic acid at the same concentration of 10μg/mL. As
shown in Figure 4B, the ΔF (F-F0) of
25-HydroxyvitaminD3 was only up to less than two-fold
higher than that of vitamin B12 when applying D3-3 aptamer as the
recognition sequence. The decrease of fluorescence intensity produced by
D3-4 aptamer were most obvious while similar fluorescence intensity
changes appeared for other three non-specific targets, demonstrating
D3-4 bound to 25-HydroxyvitaminD3 the strongest and had
only slight binding with other analogues. By comparison, D3-3 had poorer
specificity than D3-4 to 25-HydroxyvitaminD3, then D3-4
was selected as the 25-HydroxyvitaminD3-specific aptamer
with high affinity and specificity, in which the hairpin loop was the
functional domain binding to target and the double helix region was the
structural domain.
Sensitivity of the fluorescent assay
Under the above optimized conditions, the sensitivity experiment of
PG-based fluorescent assay for 25-HydroxyvitaminD3 using
D3-4 were conducted, that through obtaining fluorescence intensities at
different concentrations of 25-HydroxyvitaminD3. Figure
5 displayed the linear relationship of the fluorescence intensity (F) vs
25-HydroxyvitaminD3 concentration, presenting linear
decreases in F with increasing 25-HydroxyvitaminD3concentrations ranging from 0.04μg/mL to 0.8μg/mL. The increased
concentrations of 25-HydroxyvitaminD3 caused binding of
targets to more aptamers, so that less remaining aptamer/complementary
strand DNA duplexes were formed. The decrease in DNA duplexes and
intercalated PG lead to gradual diminution in the fluorescence
intensity. A good linear relationship between the fluorescence intensity
and the target concentration was observed in Figure 5, with a
correlation coefficient of 0.968 and a low detection limit of 0.04μg/mL.
Compared to the results of VDBA14 aptamer in relevant reference reported
by Lee et al. (2017), the truncated D3-4 aptamer performed higher
affinity and lower detection limit to
25-HydroxyvitaminD3.
Conclusions
In this study, highly sensitive fluorescent detection of
25-HydroxyvitaminD3 using truncated aptamers with
different sequences were developed. By intercepting the sequences
containing small hairpin loop and retaining double helix structural
domains of different lengths that exist in original aptamer (VDBA14), we
obtained four truncated aptamers for 25-HydroxyvitaminD3detection based on the fluorescent intensity changes of PG dye generated
upon binding to aptamer/ complementary strand-formed dsDNA duplex. After
optimization of PG concentration, we screened the truncated aptamers
through comparing the affinity and specificity to
25-HydroxyvitaminD3 target, among which D3-4 aptamer was
selected for 25-HydroxyvitaminD3 detection. The linear
dynamic range were from 0.04μg/mL to 0.8μg/mL with a correlation
coefficient of 0.968. The proposed fluorescent assay method accurately
quantified 25-HydroxyvitaminD3, attributed to high
affinity of truncated aptamer to 25-HydroxyvitaminD3 and
ultra-sensitivity of PG to trace dsDNA. Therefore, the truncated aptamer
(D3-4) with increased affinity and reduced base numbers than original
aptamer in this work is expected to be utilized for sensitive
25-HydroxyvitaminD3 detection with promising
application.
In addition, the affinities of many aptamers to complementary strands
are higher than the affinities to corresponding targets due to the long
sequences of aptamers. Thus, the applications of some methods based on
the competitive principle of target and complementary strand are
limited. In this study, affinity of aptamer to target was improved by
the truncation of original 25-HydroxyvitaminD3 aptamer,
which provided truncation reference for other long-chain aptamers.