Ion-Tagged Oligonucleotides Coupled with a Magnetic Liquid Support for the Sequence-Specific Capture of DNA

Article abstract of DOI:10.1002/anie.201703299

The isolation of specific nucleic acid sequences is a major bottleneck in molecular diagnostics. Magnetic beads/particles are typically used as solid supports for the capture of DNA targets to improve sample throughput but aggregate over time resulting in lower capture efficiency and obstruction of liquid handling devices. Herein, we describe a particle-free approach to sequence-specific DNA extraction using a magnetic liquid support and ion-tagged oligonucleotide (ITO) probes. ITO conjugates were synthesized with the highest yields ever achieved for the radical thiol-ene coupling of a substrate and oligonucleotide. In addition to distinguishing nucleotide mismatches, the ITO and magnetic liquid-based approach was more sensitive than a commercial magnetic bead-based method for the capture of target DNA from a pool of interfering genomic DNA.

Full text of DOI:10.1002/anie.201703299

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Accepted Article
Title: Ion-tagged Oligonucleotides Coupled with a Magnetic Liquid
Support for the Sequence-specific Capture of DNA
Authors: Kevin D Clark, Marcelino Varona, and Jared Anderson
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703299
Angew. Chem. 10.1002/ange.201703299
Link to VoR: http://dx.doi.org/10.1002/anie.201703299
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10.1002/anie.201703299
Angewandte Chemie International Edition
COMMUNICATION
Ion-tagged Oligonucleotides Coupled with a Magnetic Liquid
Support for the Sequence-specific Capture of DNA
Kevin D. Clark, Marcelino Varona, Jared L. Anderson*
formats. Unlike other magnetic fluids (e.g., ferrofluids) that are
particle suspensions in a carrier solvent, magnetic ionic liquids
(MILs) are neat liquids comprised of ions that respond to
magnetic fields.[16],[17] By carefully designing their chemical
structure, MILs can possess unique physicochemical properties
including hydrophobicity,[18] low viscosity,[19] and hydrolytic
stability[20] that are advantageous for biological applications in
aqueous solution.[21],[22] However, in order to exploit MILs as
supports for sequence-specific nucleic acid extraction, synthetic
oligonucleotide probes must be designed with functional groups
that facilitate dense loading onto the MIL.
Abstract: The isolation of specific nucleic acid sequences is a major
bottleneck in molecular diagnostics. Magnetic beads/particles are
typically used as solid supports for the capture of DNA targets to
improve sample throughput, but aggregate over time resulting in
lower capture efficiency and obstruction of liquid handling devices.
Here, we describe a particle-free approach to sequence-specific
DNA extraction using a magnetic liquid support and ion-tagged
oligonucleotide (ITO) probes. ITO conjugates were synthesized with
the highest yields ever achieved for the radical thiol-ene coupling of
a
substrate and oligonucleotide. In addition to distinguishing
Here we report the first synthesis and characterization of
nucleotide mismatches, the ITO and magnetic liquid-based approach
was more sensitive than a commercial magnetic bead-based method
for the capture of target DNA from a pool of interfering genomic DNA.
ion-tagged oligonucleotides (ITOs) that partition to
a
hydrophobic MIL while retaining the ability to capture specific
DNA sequences. By taking advantage of the rapid and mild
reaction conditions of thiol-ene click chemistry, a series of
imidazolium-based ion tags were appended to oligonucleotides
to profoundly influence the partitioning of the ITO probe to the
hydrophobic MIL support. Important for the practical and broad
applicability of the studied ITOs, we developed reaction
conditions that resulted in the highest yields (51-53%) ever
reported for the photoinitiated reaction between an alkene-
bearing substrate and thiolated oligonucleotide. When coupled
with real-time quantitative PCR (qPCR), we show that the MIL-
supported ITO-based approach enables the detection of target
DNA in aqueous samples containing genomic DNA interference
with greater sensitivity than a typical biotin/streptavidin magnetic
bead-based method.
Apart from storing and transferring genetic information in
biological systems, nucleic acids represent increasingly
meaningful diagnostic molecules as the complexity of the
genome continues to be unraveled. The natural ability of nucleic
acids to recognize complementary sequences through base
pairing is often leveraged through the use of synthetic
oligonucleotides for the study of clinically relevant biomarkers,[1]
gene
expression
regulation,[2]
and
single-nucleotide
polymorphisms.[3] Although modern nucleic acid sequencing and
detection technologies are capable of rapidly generating
enormous amounts of genetic data, the challenge of isolating
specific DNA/RNA targets from complex biological samples
remains a formidable bottleneck in the diagnostic workflow.
In order to obtain high sensitivity in bioanalytical assays,
nucleic acids are often captured and enriched through
hybridization with synthetic oligonucleotide probes that are
A series of ITOs were prepared from radical thiol-ene click
reactions between a 3' thiol-modified oligonucleotide (15 nt) and
allylimidazolium-based ion tags (Scheme 1) in order to study the
effect of different N-substituent groups on ITO partitioning to the
hydrophobic
MIL. Synthesis
and
characterization
of
immobilized on
a
solid particle or surface.[4] Beads,[5]
allylimidazolium salts bearing benzyl ([ABzIM+][Br−]), methyl
([AMIM+][Br−]), butyl ([ABIM+][Br−]), and octyl ([AOIM+][Br−])
substituents is provided in the Supporting Information (Figures
S1-S4). The coupling conditions required 400 nmol of alkenyl
salt and 4 nmol of thiolated oligonucleotide, likely to compensate
for sequestration of the imidazolium cation due to electrostatic
interactions with the phosphate backbone of the nucleic acid.[23]
nanoparticles,[6] or microarrays[7] bearing appropriate functional
groups ensure that oligonucleotide probes are efficiently bound
via covalent or non-covalent interactions. Magnetic beads
coated with streptavidin are particularly popular embodiments
that enable the extraction of nucleic acid targets by hybridizing
with biotinylated complements. Precise control and manipulation
of DNA/RNA-enriched beads is then accomplished by the
application of an external magnetic field, permitting the
automation of nucleic acid extraction methods for high
throughput laboratories.[8]
Scheme 1. Thiol-ene click reaction between
a
15-mer oligo and
allylimidazolium salts to generate ITOs.
An inherent limitation of sequence-specific extraction using
paramagnetic particles is the solid nature of the support.
Magnetic particles are prone to settling and aggregation which
can lead to lower extraction efficiencies,[9],[10] clogging of devices
R
N
N
Br
R
5'- TCA ACA TCA GTC TGA
oligo
N
N
R = methyl, butyl, octyl,
benzyl
oligo
systems),[11],[12]
and
require
long
S
(e.g.,
microfluidic
Br
SH
incubation/agitation times (up to 60 min) to extract a sufficient
quantity and quality of nucleic acid for downstream techniques
such as polymerase chain reaction (PCR).[13] In addition, the
binding/hybridization of nucleic acids at the solid-liquid interface
is slower (>10-fold) than in solution,[14],[15] further complicating
method development considerations.
Ideally, a magnetic liquid support that could be employed
for the magnet-based isolation of specific nucleic acid
sequences would circumvent the limitations of solid particle
40 nmol TCEP,
70/30 H2O/ACN
hv, 365 nm, N2, 1 h
ITO
The reaction products were first characterized by
denaturing polyacrylamide gel electrophoresis (PAGE, Figures
1a,b). Following PAGE, bands were excised, eluted in water
overnight, and analyzed by liquid chromatography time-of-flight
mass spectrometry (LC-TOFMS, SI for details). Mass spectra for
the ITOs (Figures 1c-f) were consistent with theoretical mass to
charge ratios (m/z, Table S1). LC-TOFMS, densitometry, and
HPLC revealed 40-45% and 51-53% yields for the [ABIM+] and
[AOIM+]-ITOs, respectively (Figures S6-S10, Table S2, and SI
for details). The yields reported here are the highest ever
[*]
K. Clark, M. Varona, Prof. J.L. Anderson
Department of Chemistry, Iowa State University, Ames, IA 50011,
USA
Email: andersoj@iastate.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201703299
Angewandte Chemie International Edition
COMMUNICATION
was evaluated using HPLC by comparing the initial amount of
ITO in solution to the amount remaining after incubation with MIL.
While 48±4% of the [AOIM+]-ITO was loaded onto the
a)
b)
−
[P66614+][Mn(hfacac)3 ] MIL, just 12±3% of the [ABIM+]-ITO and
10±3% of untagged oligo partitioned to the MIL under the same
conditions (Figures S13-S15). This suggests that ITO
hydrophobicity governs partitioning to the MIL, so the [AOIM+]-
ITO was selected for MIL-supported sequence-specific
extractions.
N
N
c)
e)
d)
f)
oligo
N
N
S
oligo
S
A
A
−4
−3
−4
−3
N
N
N
N
oligo
oligo
S
S
unreacted A
A
Denature
Anneal Capture
Release
a)
−4
−3
−4
−3
[AOIM+]-
ITO
Target
Hydrophobic
MIL
Figure 1. Denaturing PAGE of reactions between thiolated DNA and (a)
[ABIM+][Br−], [AMIM+][Br−], [ABzIM+][Br−] (lanes 1-3, respectively), and (b)
[AOIM+][Br−]. Lane 4(a) shows unmodified oligo. Mass spectra were obtained
for purified (c) [ABIM+], (d) [AMIM+], and (e) [AOIM+]-ITOs. (f) shows the mass
spectrum for the [ABzIM+]-ITO with unreacted oligo.
2500
b)
N
N
oligo
2000
1500
1000
500
0
S
A
[AOIM+]-ITO
(20-mer)
achieved for the radical coupling of a thiolated oligonucleotide
and alkene substrate.[24],[25]
Untagged 20-mer
Threshold
To study the influence of the ion tag on hybridization,
melting point (Tm) analysis was conducted for the 15-mer ITOs
with complementary strands. While the [AMIM+] and [ABIM+]-
ITOs showed no difference in Tm compared to an unmodified 15-
mer (Figure S11), a slight decrease in Tm for the [AOIM+]-ITO
and its complement was observed. Figures 2a,b show melt
curves from the [AOIM+]-ITO and an unmodified 15-mer paired
with a series of oligonucleotides with 0−2 mismatched bases. No
Tm was observed for the [AOIM+]-ITO when paired with a 2 nt
mismatch, indicating that the [AOIM+] tag further destabilizes
base mismatches when compared to unmodified probes.
The partitioning of ITOs to a hydrophobic MIL support was
investigated to determine the ITO probe that would best facilitate
NTC
0
10
20
Cycle
30
40
Figure 3. (a) The ITO-MIL strategy for sequence-specific extraction. Following
extraction and recovery of the 261 nt DNA target, (b) shows qPCR
amplification of the target when the [AOIM+]-ITO was employed (green)
compared to an unmodified complementary oligo (blue). NTC=no template
control.
Two different procedures for sequence-specific nucleic
acid isolation are commonly employed: (1) the probe sequence
is first loaded onto the support and then used to capture a
complementary target sequence or (2) the probe and target are
hybridized first, followed by addition of the support to bind the
probe-target duplex. We studied both approaches with a 15-mer
sequence complementary to the [AOIM+]-ITO using HPLC to
evaluate the amount of target extracted from solution. As shown
sequence-specific
DNA
extraction.
The
trihexyl(tetradecyl)phosphonium
manganese(II)
hexafluoroacetylacetonate ([P66614+][Mn(hfacac)3 ]) MIL was
selected as the magnetic liquid support due its hydrophobicity,
low viscosity, and paramagnetic susceptibility (synthesis in
SI).[26] The partitioning of ITO from aqueous solution to the MIL
−
Unmodified oligo
1 nt mismatch
[AOIM+]-ITO
200
180
160
140
120
100
80
300
250
200
150
100
50
a)
0 nt mismatchb)
2 nt mismatch
80
0 nt mismatch
1 nt mismatch
60
2 nt mismatch
40
20
0
0
20
40
60
Temperature (°C)
20
40
60
Temperature (°C )
80
Figure 2. Melting point analysis for (a) unmodified oligo and (b) [AOIM+]-ITO
with 0−2 nt mismatches. Tm=temperature where the maximum change in
fluorescence with respect to time occurred.
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Table 1. Comparison of the MIL-supported ITO and magnetic
bead methods for sequence-specific DNA extraction.
Constant
Sequence-specific
extraction[a]
Extraction with DNA
interference[b]
(Cq, n=3)
Support
phase
Aggregates over
time?
Responds to
Probe, support
agitation
magnetic field?
(Cq, n=6)
required?
[AOIM+]-ITO, MIL
20.33±0.96
21.92±0.92
20.82±1.10
24.07±0.31
Liquid
Solid
No
No
Yes
Yes
Biotinylated oligo,
M270 Dynabeads
Yes
Yes
[a] Extraction of 261 bp DNA (1.67 pM) in aqueous solution. [b] Aqueous sample containing 261 bp DNA (1.67 pM) spiked with 3 µg
of DNA from salmon testes. Sample volume=60 µL.
in Table S3, 50±7% of the target was extracted when the ITO
was first loaded onto the MIL compared to 25±3% when the ITO
and target were hybridized first. The difference in extraction
efficiencies is likely due to a combination of specific and non-
specific extraction of the ITO and target sequence by the MIL.
The extraction efficiency of a non-complementary 15-mer was
15±4% using either extraction approach. However, the ‘hybridize
first’ procedure would generate primarily double-stranded DNA
prior to extraction with the MIL support. Therefore, we studied
the partitioning of untagged 15 bp duplex to determine the
amount of nonspecific duplex DNA extracted by the MIL-
supported ITO. Under these conditions, <2% (n=3) of the 15 bp
DNA was extracted (Figure S16).
A qPCR assay was developed using a 261 bp DNA target
to examine the practical applicability of MIL-supported ITO-
based extraction. For this approach, a 20-mer [AOIM+]-ITO fully
complementary to a terminal segment of the 261 bp DNA was
synthesized (Figure S17). Figure 3a shows a schematic of the
MIL and ITO-based approach (details in SI). As shown in Figure
3b, qPCR amplification of the target sequence was greatly
enhanced when extracted with the [AOIM+]-ITO (Cq=20.67±1.30,
n=6) compared to using an untagged oligo (Cq=31.15±0.20,
n=3). The Cq values indicate that a 1000-fold greater amount of
DNA was extracted by the MIL-supported ITO method compared
to the approach using unmodified oligo for DNA capture (Figure
S18).
MIL exhibited much less (2±2%) nonspecific extraction. Both
methods produced similar Cq values for crude bacterial cell
lysate samples (Figure S21).
In summary, a particle-free approach for the extraction of
specific nucleic acid sequences was developed utilizing a
magnetic liquid support and ITOs. Radical thiol-ene coupling of
an oligonucleotide with an imidazolium-based ion tag resulted in
an ITO capable of partitioning to a hydrophobic MIL. When
applied for sequence-specific DNA extraction, the MIL-ITO
method demonstrated 10-fold better recovery of target DNA from
samples containing genomic DNA interference compared to a
commercially available magnetic bead-based method. Since a
magnetic liquid supports the ITO probes, we anticipate that the
ITO-based method will enable magnet-based, targeted nucleic
acid analysis using fluid handling systems that may otherwise
become obstructed by particle aggregation.
Acknowledgements
The authors acknowledge funding from Chemical Measurement
and Imaging Program at the National Science Foundation (Grant
No. CHE-1413199).
Keywords: DNA • bioconjugation • magnetic ionic liquids •
nucleic acid isolation • analytical methods
The MIL-ITO method was compared with a commercially
available magnetic bead-based method recommended for
sequence-specific DNA isolation. Target DNA (16.9 fmol) was
extracted using streptavidin-coated magnetic beads and 3’
biotinylated oligonucleotide probes according to the
manufacturer’s instructions (details in SI). As shown in Table 1,
similar amounts of target DNA were extracted by the MIL-
supported ITO and magnetic bead methods without background
DNA present. Since biological samples contain high levels of
heterogeneous sequences, we studied the effect of interfering
genomic DNA on both approaches. Samples containing target
DNA were spiked with 3 µg of DNA from salmon testes (~20
kbp) and subjected to MIL-supported ITO and magnetic bead-
based extractions. As shown in Table 1, little to no difference in
Cq values was observed for the MIL-supported ITO approach
with or without genomic DNA interference. However, higher Cq
values corresponding to a 10-fold lower amount of extracted
target were observed for the magnetic bead method with
interfering DNA (Table 1, Figure S19), likely due to nonspecific
extraction. As shown in Figure S20, the magnetic beads
extracted 20±5% of the genomic DNA whereas the hydrophobic
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
Capture
Release
qPCR Analysis
Kevin D. Clark, Marcelino Varona,
Prof. Jared L. Anderson*
2000
1500
1000
500
0
with
ion tag
Target DNA
Page No. – Page No.
w/o
ion tag
Hydrophobic
MIL
Ion-tagged Oligonucleotides Coupled
with a Magnetic Liquid Support for the
Sequence-specific Capture of DNA
Magnetic
Ionic Liquid
N
N
DNA -5'
S
10
15
20
25
30
35
40
A
Cycle
Ion-tagged Oligonucleotide (ITO)
Ion-tagged oligonucleotide probes were designed to partition to a hydrophobic
magnetic liquid, establishing the first magnet-based, liquid-supported method for
targeted DNA isolation.
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