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ABSTRACT: -Casomorphin-7 (BCM-7), a seven amino acid peptide, is released during digestion of -casein A1 variant of
milk which is speculated to be associated with certain diseases. Fifteen ssDNA aptamers having high anity toward BCM-7 were
identied from a 72 nt long random library after ten rounds of systematic evolution of ligands by exponential enrichment.
Dissociation constant values of selected aptamers were in the range of 7.7156.7 nM. Seq6 aptamer exhibited the lowest Kd
value. Nine aptamers were evaluated for their binding toward BCM-7, BCM-9A1, and BCM-9A2 peptides, and binding was
variable. SeqU5 exhibited the lowest binding with BCM-9A1 and BCM-9A2. Aptamer-coated gold nanoparticles (GNPs)
resulted in color change of GNPs in the presence of BCM-7, thereby establishing recognition of BCM-7 by aptamers. The
enzyme-linked aptamer-sorbent assay (ELASA) was evaluated as an assay of BCM-7 in biological uids. BCM-7-peroxidase
competed with BCM-7 in ELASA, performed with BCM-7 solution and BCM-7 spiked urine pretreated with urease, plasma, and
-casein digest samples.
KEYWORDS: aptamer, -casein, -casomorphin-7, SELEX, gold nanoparticles, ELASA
INTRODUCTION
Aptamers are ssDNA or RNA oligonucleotides having high
variant is speculated to be associated with certain non-
communicable diseases such as type I diabetes, coronary
heart disease, schizophrenia, and autism.13,14 A recent study has
anity toward target molecules and are selected from random
also indicated higher amounts of BCM-7 in the urine of autistic
oligonucleotide library through repetitive systematic evolution
patients,15 although the role of BCM-7 in this disease and
of ligands by exponential enrichment (SELEX) rounds.1 Each
others is still not understood. Nevertheless, the measurement of
round of SELEX results in enrichment of the oligonucleotide
BCM-7 in the blood or urine of patients may provide evidence
interacting with the target. The specicity of the oligonucleo- of a possible link between type A1 milk and these diseases.
tide is enhanced through negative and counter selection. Because of the health concerns associated with Type A1 milk,
Aptamers have been selected against numerous molecules selective breeding is being adopted in some countries to
including T-2 toxin,2 Salmonella typhimurium,3 acetamiprid,4 produce A2 milk only. At present, the PCR-based method is
botulinum neurotoxin,5 tobramycin,6 and ochratoxin A.7 The used for distinguishing between animals that will produce Type
aptamers can be used for development of sensors for A1 or Type A2 milk. The method requires isolation of DNA
tetracycline,8 estimation of kanamycin,9 and can replace from blood, somatic cells, or skin from animals16,17 and its
antibodies in immunoassays of tularemia.10 -casein A1 variant subsequent amplication by A1 or A2 allele-specic primers.
of milk (Type A1) on its breakdown in human intestine The methods for identifying BCM-7 in milk require isolation of
produces the nine amino acid peptide -casomorphin-9A1 -casein from milk and its digestion prior to HPLC.18 For
(BCM-9A1) which is further degraded into -casomorphin-7 developing a detection method, suitable anity agents which
peptide (BCM-7, a seven amino acid peptide), while -casein can recognize BCM-7 are required. Here we report the
A2 variant (Type A2 milk) on its digestion produces only - selection of 15 unique DNA aptamers that exhibited high
casomorphin-9A2 peptide (BCM-9A2), which is not further anity to BCM-7 and can be used for developing an enzyme-
degraded. The susceptibility of -casein A1 toward proteolytic linked aptamer-sorbent assay (ELASA) for BCM-7 in urine,
degradation is dierent from that of -casein A2. This is plasma, and -casein digest.
because of the dierence in one amino acid at the 67th position
of -casein. Proline is present at the 67th position in A2 variant,
whereas this amino acid is substituted by histidine in A1
MATERIALS AND METHODS
Safety. Gold nanoparticles, magnetic beads, and chemicals such as
variant.11 Histidine at the 67th position in the A1 variant allows ethidium bromide were used during the experiment. However, all the
cleavage of the peptide bond between the 66th and 67th amino materials were disposed of safely according to Institute regulations.
acid residues by intestinal protease, which results in formation Human and Animal Rights Issues. The work is in vitro in nature
of BCM-7.12 BCM-7 from the intestine can be transported to and does not include any human or animal trials.
blood in infants and people suering from autistic and
schizophrenic disorders having high intestinal permeability Received: October 2, 2014
and low activity of Dpp-iv (Dipeptidyl peptidase-4) enzyme. In Revised: February 23, 2015
blood, BCM-7 is further degraded to -casomorphin-5 Accepted: February 25, 2015
(BCM-5) by Dpp-iv enzyme. Milk containing -casein A1
Conjugation of BCM-7, BCM-5, BCM-9A1, and BCM-9A2 SELEX. An amplied ssDNA library (10 g) was incubated with
Peptides to Magnetic Beads. BCM-7, BCM-5, BCM-9A1, and BCM-7 conjugated beads (2 108) in 100 L of binding buer for 1 h
BCM-9A2 peptides (Technoconcept) were conjugated to tosyl at 28 C with intermittent tapping of tubes to prevent settling of
activated Dynabeads M-280 (Invitrogen) as per manufacturer magnetic beads. Then, the beads were washed with 100 L of binding
protocol. A 100 mM borate buer was prepared by adding 6.18 g of buer to remove unbound ssDNA, while bound ssDNA was eluted
boric acid in 800 mL of water. The pH was adjusted to 7.8 by 1 N with elution buer (20 mM Tris HCl, 10.0 mM EDTA, 3.5 M Urea,
NaOH; the nal volume of the solution was made to be 1000 mL by and 0.02% Tween 20, pH 8.0) at 80 C for 10 min. Ten rounds of
the addition of water. A sample of 2 108 beads were washed four SELEX were performed to enrich target-specic oligonucleotides
times with 100 mM borate buer (pH 7.8). To the washed beads were (aptamers). To enhance the specicity of the selected oligonucleo-
added 100 L of peptide (1 mg/mL in water); 50 L of 100 mM tides, negative selector and counter selectors (BCM-5, BCM-9A1, and
borate buer, pH 7.8; and 100 L of 3.0 M ammonium sulfate BCM-9A2 coated magnetic beads) were used. In negative selection,
(prepared in borate buer, pH 7.8). The contents were incubated for tosyl activated magnetic beads were blocked by 0.5% BSA (prepared in
20 h at 37 C. The unconjugated peptides were removed by washing PBS) for 1 h at 37 C. In each round of SELEX, ssDNA eluted from
the beads with borate buer. The remaining sites on the beads were the last elution was amplied. Amplication conditions used are as
blocked by immersing beads in 0.5% bovine serum albumin (BSA) follows: an initial heat activation step at 95 C for 3 min and 17 cycles
prepared in phosphate buer saline (PBS), pH 7.4, for 1 h at 37 C. of 95 C for 14 s, 55 C for 23 s, 72 C for 17 s, and an extension step
After the blocking solution was removed, peptide conjugated beads of 2 min at 72 C. The concentration of PCR product was determined
were suspended in 0.1% BSA-PBS, pH 7.4, stored at 4 C, and used by nanophotometer and was digested by addition of 10U
within 15 days. exonuclease for every 2.0 g of amplied product. The digested
Amplication of ssDNA Library. A 500 ng sample of 72 nt long PCR product was puried by using Qiax II gel extraction kit, snap
ssDNA (MW = 22 067 Da) library19 (Integrative DNA technology, cooled, and used for the next round of SELEX. The conditions in
IDT) was amplied in 5 parallel reactions by PCR. This will essentially various rounds of SELEX are provided in Table 1.
have (500 109)(6.023 1023)/22 067 = 1.36 1013 molecules. Enrichment of BCM-7 specic aptamer after each round of SELEX
Total randomness possible from 36 variable nucleotides in the 72 nt. was evaluated by amplication of 1.0 L of aliquot taken from each
long library will be 436 = 4.7 1021. However, the maximum round of washings and elutions. The PCR reaction conditions used
randomness from a 500 ng library will be limited to 1013. Each reaction were the same as those mentioned above. The PCR product was
mixture was composed of 50 mM (NH4)2SO4, 100 mM Tris-HCl, 2.0 conrmed by resolving on 3% agarose gel electrophoresis. The amount
mM MgCl2, 0.2 mM dNTPs, 100 ng of ssDNA library, 1.0 M each of of DNA was calculated by gel analysis software (SynGene, Synoptis).
forward primer (5-ATCCGTCACACCTGCTCT-3) and phosphory- The aptamers obtained after the last round of SELEX were amplied
lated reverse primer (5-P-ATACGGGAGCCAACACCA-3), and 1.5 by using forward primer (5-ATCCGTCACACCTGCTCT-3) and
U Taq DNA polymerase in a volume of 50 L. The amplication reverse primer (5-ATACGGGAGCCAACACCA-3). The PCR
conditions used are as follows: an initial heat activation step at 95 C product was inserted into pGEM T cloning vector (Promega). The
for 5 min and 30 cycles of 95 C for 1 min, 55 C for 1 min, 72 C for recombinant vector was cloned into Escherichia coli (DH5). A total of
1 min, and an extension step of 2 min at 72 C. Amplication of the 41 clones were selected for sequencing (SciGenom).
library was conrmed by resolving PCR product on 3% agarose gel Determination of Dissociation Constant. Prior to use, BCM-7
electrophoresis. conjugated magnetic beads were washed and suspended in binding
Conversion of dsDNA into ssDNA. Amplied PCR product buer. A 100 L sample of 5325 nM, 5(6)-carboxyuorescein
without further purication was digested by exonuclease20 labeled aptamer (IDT) (prepared in binding buer) was mixed with 50
(Fermentas) and digestion reaction was monitored on 3% agarose L of BCM-7 conjugated beads (1 108) for 1 h at 28 C. Unbound
gel electrophoresis. After 100 min of digestion, the reaction was aptamers were collected by ve quick successive washing with binding
stopped by inactivating exonuclease, which was achieved by heating buer of 100 L each. Bound aptamers were eluted with ve
at 85 C for 10 min. The product was puried by Qiax II gel extraction successive treatments of beads with elution buer of 100 L each at 80
kit (Qiagen). The preparation (pure DNA) was stored at 20 C until C for 10 min.22 The unbound and bound aptamers were separately
further use. On the day of SELEX, the contents were thawed and pooled. Labeled aptamers were measured at 494 nm excitation and
diluted with 100 L of binding buer (20 mM Tris HCl, 100 mM 520 nm emission wavelengths (Varian, Cary Eclipse Fluorescence
NaCl, 2.0 mM MgCl2, 5.0 mM KCl, 1.0 mM CaCl2, and 0.02% Tween Spectrophotometer). The dissociation constant (Kd) was calculated by
20, pH 7.6). The undigested reverse strands were further separated by nonlinear regression analysis using Graph Pad Prism 5 software.
snap cooling, which was achieved by heating of puried product at 90 Binding Selectivity of BCM-7 Apatmers. Selected aptamers
C for 15 min and immediately placing on ice for 5 min.21 were tested for their ability to bind with BCM-7/BCM-9A1/BCM-
B DOI: 10.1021/acs.jafc.5b00007
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article
D
a
group
bases.)
group 6
group 5
group 4
group 3
group 2
group 1
motif 5
motif 4
motif 3
motif 2
motif 1
motif
5CAGGTGTG 3
no conserved motif
5 CGGNNNCCG 3
5 ACACANTGTGT 3
5GGAGCCAACACC 3
5CTCTATTCGAGAG 3
Table 3. Groups of Aptamers
seq10(2)
seqU3(2)
seqU4(4)
seqU5(5)
seqU4(4)
seq9(2) and
seq1(5) and
seqU2(2) and
and seq8(1)
seq6(2), and
seqU1(2), and
seq2(3), seq3(1),
seq1(5), seq6(2),
seq1(5), seq4(3),
seq5(1), seq7(3),
name of aptamersa
and counter selector peptides (BCM-9A1 and BCM-9A2).
41.8523.11
37.5337.66
7.69156.66
7.69141.26
Kd range (nM)
DOI: 10.1021/acs.jafc.5b00007
Figure 1. Percentage of bound aptamers for target peptide (BCM-7)
activity and BCM-7 concentration. The IC50 value was 250 ng/
mL for BCM-7 (Figure 4a).
Cross recognition of selected nine aptamers to BCM-5,
BCM-9A1, and BCM-9A2 peptides was also checked. In a 96-
well plate format, BCM-7-peroxidase competed with BCM-5,
BCM-7, BCM-9A1, or BCM-9A2. The extent of binding of
these peptides is reected in the extent of the drop in
absorbance. For example, seqU5 aptamer has failed to
recognize all the peptides including BCM-7. Seq7 has cross
reacted with BCM-5, BCM-9A1, and BCM-9A2. Seq3, seq4,
seq6, and seqU4 have shown lower cross-recognition among
the nine aptamers tested (Figure 4b).
The ELASA method was used to evaluate whether aptamers
could recognize spiked BCM-7 in biological uids. Urine
contains urea which may interfere in recognition of BCM-7 by
aptamer; therefore, the urine sample was treated with urease
before its use in ELASA. BCM-7-peroxidase competed with
spiked BCM-7 present in urine, plasma, and -casein digest
samples as indicated by lowered absorbance with increasing
concentration of BCM-7 (Figure 5). Results also suggest that
some component of urine and plasma interferes in the assay
(see absorbance value in corresponding control and blank
Figure 3. Color change in gold nanoparticles (GNPs): (a) NaCl samples) (Figure 5).
addition at indicated concentrations (mM) to GNPs, (b) NaCl
addition at indicated concentrations in mM to GNPs conjugated to
aptamer (seqU4), and (c) color change in GNPs conjugated with
DISCUSSION
aptamer (seqU4) on incubation with dierent concentrations (g/ BCM-7 has been linked with certain noncommunicable diseases
mL) of BCM-7 and 350 mM NaCl. such as type I diabetes, coronary heart disease, schizophrenia,
and autism; suitable ligands which can recognize BCM-7 are
aptamers (seq1, seq7, seq10, seqU4, and seqU5) was allowed to required for method development. This paper describes in vitro
bind to the plate using biotinavidin interaction. BCM-7 and selection of aptamers that bind to BCM-7 with high anity.
BCM-7 peroxidase conjugate competed for limited amount of Out of 15 aptamers, ve aptamers are generated with forward
aptamer. There was an inverse relationship between peroxidase primer while ten aptamers start with reverse primer. This
Figure 4. ELASA for BCM-7. The extent of displacement of the BCM-7 peroxidase conjugate by (a) BCM-7 peptide at dierent concentration and
(b) BCM-7, BCM-5, BCM-9A1, and BCM-9A2 at xed concentration.
E DOI: 10.1021/acs.jafc.5b00007
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article
Figure 5. ELASA for BCM-7 in urine, plasma, and -casein digest. Control and blank represent peroxidase activity in the absence of BCM-7 in
binding buer, urine, plasma, or -casein digest, respectively.
clearly indicates that exonuclease was not able to fully digest base method for assaying BCM-7 from urine requires prior
the dsDNA. Available protocol for digestion of dsDNA by extraction for determination of BCM-7 concentration.15
exonuclease requires prior purication of PCR product.
However, there is a loss in yield that occurs during purication.
In the present work, PCR product was digested with
*
ASSOCIATED CONTENT
S Supporting Information
exonuclease without prior purication. This results in Mfold structure of aptamers and photos of aptamers showing
incomplete digestion. In fact, 13% dsDNA (GelQuant color change at dierent concentrations of BCM-7. This
software) was found in DNA-digest by exonuclease, based material is available free of charge via the Internet at http://
on the intensity of dsDNA and ssDNA bands after their pubs.acs.org.
separation on agarose gel electrophoresis. Thus, the remaining
dsDNA was converted to ssDNA by snap cool method. AUTHOR INFORMATION
The mean Kd values of forward strand aptamer (mean Kd = Corresponding Author
42.0 nM) and reverse strand aptamers (mean Kd = 50.2 nM) *Phone: +91184-2259129. E-mail: ys_rajput@redimail.com.
suggest that both groups of aptamers have similar binding
anity for the target. The Kd values of repeat-aptamers were Notes
The authors declare no competing nancial interest.
also compared with those of nonrepeat aptamers, and it was
found that the mean Kd values of repeat-aptamers (mean Kd =
42.0 nM) were similar to that of nonrepeat aptamers (mean Kd ACKNOWLEDGMENTS
= 49.5 nM). Research funding and fellowship was provided by National
Nine aptamers (seq1, seq10, seq7, seqU5, seq3, seq4, seq6, Dairy Research Institute, Karnal-132001, India.
seqU2, and seqU4) were used for comparing their specicity in
two dierent experimental designs (Figures 1 and 4b). The
results suggest that the extent of cross reaction of aptamers with
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F DOI: 10.1021/acs.jafc.5b00007
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article
G DOI: 10.1021/acs.jafc.5b00007
J. Agric. Food Chem. XXXX, XXX, XXXXXX