Fluorogenic Templated Reaction Cascades for RNA Detection

Article abstract of DOI:10.1021/jacs.7b00466

Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of in

Full text of DOI:10.1021/jacs.7b00466

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Article
Fluorogenic Templated Reaction Cascades for RNA Detection
Willem A. Velema, and Eric T. Kool
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00466 • Publication Date (Web): 27 Mar 2017
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Fluorogenic Templated Reaction Cascades for RNA Detection
Willem A. Velema and Eric T. Kool*
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Department of Chemistry, Stanford University, Stanford, CA, 94305, USA
ABSTRACT: Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological
states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal
cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal
amplification, which hinders the detection of small amounts of genetic material. To address this limitation, we tested a
new strategy for attaining higher-order signal amplification, in which a target sequence templates a chemical ligation and
the product of this reaction is in turn detected with a second templated reaction. The method is nonenzymatic, isother-
mal and fluorogenic, allowing the direct detection of nucleic acids in complex matrices. Using this approach, as little as
500 attomoles (10 pM) could be detected with single nucleotide resolution. In a test of selectivity, single nucleotide substi-
tutions and deletions could successfully be detected, including a deletion that is associated with tetracycline resistance in
Helicobacter pylori. Compatibility with biological matrices was demonstrated by the direct detection of rRNA in bacterial
lysate. Imaging and detection of target sequences on a solid support further illustrates the potential of the new approach
for
high-throughput
analysis.
INTRODUCTION
reaction. The product of this ligation can itself template a
second reaction, resulting in exponential amplification.
To date, such quadratic and exponential amplifying
systems have not been developed for the nonenzymatic
and direct detection of cellular nucleic acids. We
hypothesized that use of carefully coupled templated
chemistries together with fluorogenic signaling might be
applied to detect small quantities of genetic material.
The rapid detection and imaging of minute amounts of
nucleic acids is an ongoing challenge that holds great
promise for application in biology and medicine.1–6 DNAs
and RNAs contain valuable information about
pathological conditions and disease progression and can
be powerful biomarkers for diagnostics including cancer
pathogenesis,3
identifying
infections2
and
drug
resistance.7 However, the direct detection of nucleic acids
in biological samples without sample preparation and
target isolation remains challenging.8
Recent technologies like nanoflares,9 hybridization
chain
reactions,10
molecular
beacons,11
strand
displacement12,13 and nucleic acid-templated reactions14,15
are aimed at addressing these issues, and offer turn-on
signaling without enzymes. However, the linear
amplification of these strategies results in limited signal
gain. In particular, fluorogenic RNA- and DNA-templated
reactions have gained popularity,16–20 because the bio-
orthogonality and isothermal nature of these approaches
allow for the direct detection of nucleic acids in biological
samples without the need for any work-up or target
isolation. To date, however, the linear amplification of
such methods limits their sensitivity.8
Figure 1. Schematic representation of the fluorogenic ampli-
fied cascaded templated reaction (FACTR). The first reaction
(QUAL, red probes) is a chemical ligation that forms a desta-
bilized product, which dissociates and in turn templates the
second reaction (QSTAR, green probes). A TPP-bearing
probe reduces an azido-ether linker, causing the release of a
fluorescent quencher, resulting in an increase in emission.
During the course of the reactions, more product template is
being formed in the QUAL reaction, which is available to
catalyze more QSTAR reaction, resulting in quadratic signal
generation. LG= leaving group; TPP=triphenylphosphine;
QUAL= quenched autoligation; QSTAR= quenched
Staudinger azidoether release.
RNA and DNA in nature evolved to support
amplification
beyond
linear
dimensions.
The
extraordinary molecular recognition properties of nucleic
acids can be exploited to propagate higher-order
replication and signal generation.8,21–23 Several in vitro self-
replicating and cross-replicating systems that rely on
base-pairing have been developed, including RNA
enzymes24–26 and nucleic acid- templated ligation
reactions.27–29 For example, in a study by Gibbs-Davis,29,30
a
target sequence templates an enzymatic ligation
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7.0, 10 mM MgCl2 at 30 °C for 20 h unless indicated differ-
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Here we describe the coupling of two nucleic acid-
templated reactions to attain quadratic signal formation.
Both templated reactions are nonenzymatic and
isothermal, and the initiation of the replication process
depends solely on the presence of a target sequence. The
fluorogenic amplified cascaded templated reactions
(FACTR) enable direct detection of nucleic acids in
complex matrices. Using this approach, as little as 500
attomoles of a target could be detected with single
nucleotide resolution.
ently. This was added to 200 nM QSTAR-DNA, 400 nM
TPP-DNA in buffer. The parallel reactions were carried
out with 1 µM UL-DNA and Thio-DNA, 200 nM QSTAR-
DNA, 400 nM TPP-DNA and template concentrations as
indicated in buffer at 30 °C. Fluorescence emission was
observed on a Fluorolog 3-11 instrument (Jobin Yvon-
SPEX). λex=497 nm and λem=519 nm. Graphs were
smoothed by adjacent averaging.
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Detection in lysate. Escherichia coli (E. coli) K12 (ATCC
107988) cells were grown overnight at 37 °C in LB media
(10 mL) to an OD600 of ~ 2.5. Cultures were centrifuged
and the supernatant discarded. The pellet was resuspend-
ed in lysis buffer and RNAse OUT (Invitrogen) was added
to a final concentration of 1u/µL. Cell disruption beads
(0.1 mm) were added and the suspension was vortexed at
4 °C for 10 min. The suspension was centrifuged and the
supernatant was diluted 100x in tris-borate buffer and
stored at -80 °C until use. 5 µM UL-DNA and Thio-DNA
were incubated in lysate for 16 h at 30 °C. This was added
to 200 nM QSTAR-DNA and 400 nM TPP-DNA in buffer
and fluorescence emission was observed.
On-bead detection. Q-STAR probes were immobilized
on Oligo-Affinity Support (PS) (Glen Research) and a
hexaethylene glycol linker was incorporated between the
support and oligonucleotides to assure spacing. QSTAR
probes were synthesized on the support and included a 5’
amino modifier 5 (Glen Research). QSTAR probes were
modified on the support post-synthesis to attach the az-
ido-ether dabsyl quencher and washed thoroughly. 5 µM
UL-DNA and Thio-DNA and 100 nM WT template in
buffer were incubated for 20 h at 30 °C. This solution was
added to the QSTAR-modified beads and 5 µM TPP-DNA
and the beads were imaged on a Nikon Eclipse 80i epiflu-
orescence microscope equipped with a Nikon Plan Fluor
10x objective and a QIClick digital CCD camera.
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By exploiting inherent selectivity of one of the
templated reactions we could detect a single nucleotide
deletion that is associated with tetracycline resistance in
Helicobacter pylori (H. pylori). Moreover,
a single
nucleotide substitution was efficiently detected by
employing a second set of probes that complement the
substitution. The compatibility of the coupled templated
reactions with biological matrices was established by
targeting 16S rRNA in bacterial lysate, which resulted in a
selective and robust fluorescence signal. Furthermore, on-
bead detection of target sequences was achieved,
demonstrating the potential for high-throughput analysis
by bead imaging.
MATERIALS AND METHODS
Synthesis. Details on the synthesis of the DNA modifying
groups are given in the Supporting Information. QSTAR
and QUAL modifying groups were prepared as previously
reported.31,32
Oligonucleotide probes. Oligonucleotides and DNA-
conjugates were prepared on an ABI 392 synthesizer on a 1
µmol scale using standard phosphoramidite chemistry.
All reagents and columns were purchased from Glen Re-
search Corporation. DNA strands were deprotected and
cleaved with concentrated ammonium hydroxide at 55 °C
for 16 h. Ultramild protected DNA strands were depro-
tected and cleaved with 0.05 M K2CO3 in methanol at
room temperature for 5 h. Oligonucleotides were purified
using RP-HPLC and eluted with MeCN/0.1 M TEAA buff-
er. Unmodified DNAs were purchased from Integrated
DNA Technologies, Inc. Universal linker probes and 3’
thiophosphate oligodeoxynucleotides were prepared as
described previously.32 QSTAR and TPP-probes were ob-
tained by post-DNA synthesis modification as reported.31
QUAL probes turnover.
Turnover of the QUAL probes in the first amplification
step was determined with analytical RP-HPLC (Shimadzu
LC-10AD and SPD-M10A Diode Array Detector equipped
with an XBridge Oligonucleotide BEH C18 column) and
eluted with ACN/0.1 M TEAA buffer. The peak area was
determined with Shimadzu EZstart 7.4 software and was
converted into the amount of product formed using a
calibration curve (Fig. S2). The turnover number was de-
fined as the number of formed products per template
strand.
RESULTS
Design of coupled templated reactions. To potentially
achieve quadratic signal formation, we explored whether
two templated reactions could be combined (Fig. 1). The
challenge of our two reaction design is to avoid
interference and self-inhibition. The first reaction chosen
was a chemical autoligation32 (QUAL) that results in the
formation of a linked but hybridization-destabilized
product (Fig. 2A). Upon binding of the two QUAL probes
to a target sequence, the nucleophilic phosphorothioate
group of one probe (Thio-DNA) attacks the electrophilic
carbon of a butyl group on the neighboring probe (UL-
DNA). The resulting butyl linker was shown to be
destabilizing and enhances dissociation of the ligated
product from the target, enabling turnover.32 We
envisioned that this first linked product might then act as
the template for a second reaction, which we chose to be
a Quenched Staudinger Azidoether Reduction (QSTAR,
Fig. 3A).31 One QSTAR probe bears a triphenylphosphine
(TPP) group that will reduce an azide on the neighboring
probe when bound adjacent on the template. This results
in the release of a dabsyl quencher, causing an increase in
Coupled templated reactions. The coupled templated
reactions in series were carried out with 5 µM Universal
Linker DNA (UL-DNA) and Thio-DNA and template con-
centrations as indicated in 70 mM Tris-Borate buffer pH =
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fluorescence emission of the fluorescein label also
attached to the probe (Fig. 1).
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Both reactions are nonenzymatic, isothermal, and
are expected to be chemically orthogonal with each other.
During the course of the first reaction, increasing
amounts of ligated product strands will be formed that
will then be available, in principle, as templates for the
QSTAR reaction. If successful, this is expected to result in
a quadratic conversion of quenched substrate probes into
fluorescent products, since the QSTAR reaction itself was
shown to exhibit substantial turnover.31 One unknown in
this FACTR strategy was whether the QUAL ligation
product could serve as an effective template for QSTAR,
since the two halves of the ligated probe are separated by
an unnatural butyl linker, thus potentially interfering
with the subsequent templated reaction by separating
them with unnatural structure.
Additionally, we envisioned a potential mechanism
for achieving high selectivity for target detection: in
previous work it was shown that the QUAL reaction
exhibits significant turnover only when a nucleobase is
positioned opposite to the destabilizing linker on the
template strand (Fig. 2a).32 This could potentially be
exploited to detect deletions: when the QUAL reaction
does not turn over, there will be no template available to
catalyze the QSTAR reaction and no fluorescence increase
will be observed. A single nucleotide deletion at position
733 of Helicobacter pylori (H. pylori) 16S rRNA is
associated with tetracycline resistance.33 Probe sequences
were chosen to flank this position allowing for
discrimination between wild-type and tetracycline-
resistant H. pylori 16S rRNA (Fig. 2) with the FACTR. All
probes were synthesized as previously described31,32 using
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Figure 2. Performance of the QUAL reaction, which ligates
reactive probes on an RNA or DNA template. A) Probe de-
sign and target sequences. MUT template has a single point
deletion compared to WT template. B) Effect of target se-
quence and concentration on turnover. Reactions were car-
ried out in 70 mM Tris-Borate buffer pH = 7.0, 10 mM MgCl2,
at 30 °C, with 5 µM UL-DNA and Thio-DNA with varying
amounts of target template for 20 h. Error bars show stand-
ard deviations from triplicate measurements. LG=leaving
group.
phosphoramidite
chemistry
and
post-synthesis
modification for TPP-DNA and QSTAR-DNA probes.
Performance of individual QUAL and QSTAR re-
actions. To assess the performance of the first-stage tem-
plated reaction (QUAL) in the H. pylori sequence context,
product formation was monitored by RP-HPLC (see Fig.
S1). QUAL probes were incubated with wild-type (WT)
and resistant (MUT) template in tris-borate buffer. Reac-
tions proceeded in high yields, and a significantly higher
turnover number of 20 was found for the WT template,
compared to only 4 for the MUT template at a concentra-
tion of 1 nM (Fig. 2B). This is in concurrence with earlier
work that showed a nucleobase opposite to the universal
linker is required for enhanced turnover and supports the
hypothesis that this approach might be exploited to de-
tect single nucleotide deletions. At a higher template con-
centration of 100 nM the turnover number drops signifi-
cantly as expected due to the relative smaller excess of
QUAL probes.32,34 Turnover numbers are typically low to
moderate for ligation reactions,32,34 but the overall
turnover is expected to be significantly improved when
combined with a second templated reaction.
The probes for the QSTAR reaction consist of a 3’
TPP bearing ODN (TPP-DNA) and an ODN that is
equipped with a fluorescein label and a 5’ azidoether link-
er that carries a dabsyl quencher. The QSTAR probes are
designed to target the product of the QUAL reaction. As
mentioned above, this product contains a butyl linker
that could potentially interfere with the efficiency of the
QSTAR reaction, as the reacting probes are separated by
the added intervening bonds. To examine this, a syntheti-
cally prepared (see SI) butyl linker-bearing ODN was
tested as a template for the QSTAR reaction, alongside
the analogous natural ODN. The data showed clearly (Fig.
3B) that the presence of the butyl linker has no deleteri-
ous effect on the rate of the QSTAR reaction despite the
intervening linker.
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Figure 3. Performance of QSTAR reaction with natural and
butyl linker-containing DNA. A) QSTAR probe design and
template sequence with and without butyl-linker. When in
close proximity, the TPP will reduce the azide functionality,
resulting in cleavage of the azidoether linker and release of
the quencher. F= fluorescein; Q= quencher (dabsyl); TPP=
triphenylphosphine. B) Time course of fluorescent increase
with both templates under equimolar conditions. 200 nM
template 3 or 4, 200 nM QSTAR-DNA and 400 nM TPP-DNA
in 70 mM Tris-Borate buffer pH = 7.0, 10 mM MgCl2 at 30 °C.
λex=497 nm and λem=519 nm.
Coupled Nucleic Acid Templated Reactions. Ini-
tial studies on the performance of the FACTR were con-
ducted by running the two templated reactions in series.
Relatively high concentrations of QUAL probes (5 µM)
were incubated with 100 nM of WT or no template in tris-
borate buffer for 20 h at room temperature. When TPP-
DNA (400 nM) and QSTAR-DNA (200 nM) were then
subsequently added to this solution, a robust increase in
fluorescence emission was observed in the WT template
sample and not
in the control (see Fig. S3), indicating that product tem-
plate had formed during the QUAL reaction and could be
detected with QSTAR probes. To optimize the conditions,
the reactions were carried out at varied temperatures and
the initial rates of fluorescent increase were compared
(Fig. 4a). Reactions at 30 °C were found to have the high-
est rate and therefore this temperature was chosen for all
further experiments. The calculated melting temperatures
(Tm’s) of UL-DNA and Thio-DNA to the target are
around 37 °C. The ligation reaction is relatively slow,
which is possibly why temperatures below the Tm result
in a higher initial rate. This is in accordance with earlier
observations.32
Figure 4. Sequential coupling of templated reactions. A)
Analysis of initial fluorescence rate at different temperatures
with 100 nM WT target. The rate was determined by averag-
ing the slope from 15-30 min. Error bars represent measure-
ments in triplicate. B) Amplified fluorescent signal under sub
stoichiometric amounts of WT template. C) Time course of
fluorescence with 100 nM of template 1, 2, 3 or no template.
The coupled templated reactions were carried out with 5 µM
UL-DNA and Thio-DNA and template concentrations as in-
dicated for 20 h in 70 mM Tris-Borate buffer pH = 7.0 10 mM
MgCl2 at 30 °C. This was added to 200 nM QSTAR-DNA, 400
nM TPP-DNA in buffer. λex=497 nm and λem=519 nm.
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template that has a single point deletion that is associated
with tetracycline resistance in H. pylori.33 As is apparent
from the data (Fig. 4C), there is a large difference in
fluorescence increase between WT and MUT template.
This can be attributed to decreased destabilization when
there is no nucleobase opposite to the butyl linker in
duplex DNA. These results show that the FACTR is
efficient in the discrimination of single point deletions.
Although QSTAR probes have been shown to be highly
sensitive to point mutations,31 we know of no previous use
of templated reactions to detect single nucleotide
deletions. To broaden the applicability of the method to
other single nucleotide polymorphisms (SNPs) we tested
the possibility of detecting substitutions. When template
3 containing a single base mutation was used in the
FACTR, a considerably lower fluorescence signal was
obtained compared to the WT template 1 (Fig. 4C). QUAL
and QSTAR probes were then prepared that complement
the single nucleotide substitution template 3; when
applied in the FACTR, fluorescence was fully restored
(Fig. 4C). This indicates that the new method can be
applied for detection of multiple classes of SNPs.
It might be advantageous in certain situations to run
the two templated reactions in parallel, i.e. simultaneous-
ly in one tube. All probes and target are then added at the
same time, which simplifies sample handling. To assess
the performance of the FACTR when conducted in paral-
lel, target template (100 nM), UL-DNA, Thio-DNA (both 1
µM), QSTAR-DNA (200 nM) and TPP-DNA (400 nM)
were incubated simultaneously for 20 h at 30 °C. Next,
fluorescence emission spectra of the samples with and
without template were taken. A clear difference in emis-
sion was observed between the sample with and without
WT template (Fig. 5a), showing that both templated reac-
tions can be performed simultaneously in the same solu-
tion. When the fluorescence emission was monitored over
time, a sigmoidal-shaped curve was observed (Fig. 5B), as
expected for quadratically amplifying systems.8 The limit
of detection (Fig. S4) was relatively high when compared
to the FACTR in series. This is possibly due to back-
ground signal from untemplated hydrolysis of QSTAR-
DNA. This might be overcome in future experiments by
employing doubly quenched QSTAR-DNA.35
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Figure 5. Simultaneous coupling of templated reactions. A)
Fluorescence emission spectra of the coupled templated re-
actions when carried out simultaneously after 20 h incuba-
tion with 100 nM of WT, MUT or no template. B) Sigmoidal
amplification by the simultaneous templated reactions with
100 nM of WT template. The graph is background corrected
by subtracting the fluorescence from a blank reaction lacking
template. The coupled templated reactions were carried out
with 1 µM UL-DNA and Thio-DNA, 200 nM QSTAR-DNA,
400 nM TPP-DNA and template concentrations as indicated
in 70 mM Tris-Borate buffer pH = 7.0 10 mM MgCl2 at 30 °C.
λex=497 nm and λem=519 nm.
Detection of rRNA in biological samples. We set
out to target 16S rRNA in bacterial lysate to explore the
utility of this method for detection of genetic sequences
in biological samples. Applying nucleic acid-templated
reactions for the detection of certain genetic sequences
holds great promise for diagnosing infections7 and other
pathological conditions3 as well as studying biological
processes such as cell differentiation.36
Our initial set of probes target 16S rRNA in H. pylori,
which is homologous to Escherichia coli (E.coli) 16S rRNA
at the targeted site.37 Therefore, experiments were con-
ducted with E. coli as a safer alternative to H. pylori. Bac-
teria were grown overnight to an OD600 of ~ 2.5 and lysed
by bead beating (see SI). QUAL probes were added to the
supernatant and incubated for 20 h at 30 °C. Next, the
QSTAR probes were added to the solution and a robust
increase in fluorescence was observed (Fig. 6A). As a con-
One outstanding challenge is to be able to detect
small amounts of nucleic acid. 10-fold dilutions of WT
template were subjected to the nucleic-acid templated
network to determine the limit of detection. At a concen-
tration of 10 pM (500 amol) WT template, a significant
signal could still be detected over background. At lower
concentrations, signal was not distinguishable from back-
ground. The results show that rate of fluorescence in-
crease is dependent on the amount of target template.
Therefore, a calibration curve may allow quantification of
template between 1 and 10 pM.
To test the utility of this approach for the detection of
single nucleotide deletions, probes were incubated with
WT template, representing H. pylori 16S rRNA, and MUT
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trol, Thio-DNA and QSTAR-DNA that target 16S rRNA 50 QSTAR-DNA. The fluorescent product of this reaction
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nucleotides downstream from the initial sequence were
synthesized (see SI). In this case, control UL-DNA and
control Thio-DNA are spatially separated and should
therefore not ligate to form the template for the QSTAR
reaction. Indeed, an absence of fluorescence was observed
when these probes were employed in bacterial lysate (Fig.
6A). This strongly indicates that the increase in emission
when the neighboring probes are used stems from the
interaction between these probes and the 16S rRNA target
sequence. Thus we conclude that FACTR is efficient in
detecting specific rRNA sequences in bacterial lysate.
cascade is retained on the surface and would result in a
green emissive bead.
To test this, the fluorogenic QSTAR-DNA probes were
immobilized on 130-micron PEG-polystyrene beads (see SI
for methods). Reactions were carried out with 5 µM ULꢀ
DNA and ThioꢀDNA and 100 nM WT template in buffer
and were incubated for 20 h. Next, the solution was added
to the QSTARꢀmodified beads and 5 µM TPPꢀDNA and
the results were monitored by fluorescence microscopy. A
clear difference in fluorescence signal was observed be-
tween the reactions that were conducted with and with-
out WT template (Fig 6B and 6C). These preliminary ex-
periments show that the nucleic acid-templated network
can indeed be used to detect genetic sequences on a solid
support and can potentially be employed for high-
throughput genetic detection.
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DISCUSSION AND CONCLUSIONS
Our data demonstrate that the molecular recognition
properties of nucleic acids can be exploited to devise a
network of nonenzymatic templated reactions that can
achieve quadratic signal formation. Two nucleic-acid
templated reactions were combined, in which the first
reaction yielded a ligated product that could catalyze the
second reaction, which in turn produced a fluorescent
product. Our results show that we could employ the nu-
cleic-acid templated network to detect target templates in
a concentration-dependent manner and a target quantity
of 500 amol (10 pM) could still be detected significantly
over background. We know of no previous reports of such
cascaded nonenzymatic and fluorogenic methods for nu-
cleic acid detection with single nucleotide resolution. In
recent work by the group of Gibbs-Davis,29 a self-
replicating nucleic-acid templated reaction was developed
that relied on destabilization by an abasic lesion. This
elegant approach showed sigmoidal amplification, but
relied on enzymatic ligation and was not assessed on di-
rect detection in complex matrices. Recent work by our
laboratory40 combined rolling circle amplification (RCA)
with a nucleic-acid templated reaction to attain quadratic
amplification. That method contained a convenient fluo-
rescent readout, but still relied on enzyme activity to per-
form RCA, and also relied on a denaturing step.
By exploiting the destabilizing properties of auto-
ligated probes in our FACTR approach, a single-point
deletion that is associated with tetracycline resistance in
H. pylori could be detected. A considerable difference in
fluorescence increase was observed over the wild-type
sequence. This could potentially be useful for the diagno-
sis of pathological conditions that are associated with
single point deletions such as antibiotic-resistant infec-
tions33 and genetic disorders like Tay-Sachs disease.41 Alt-
hough previous template chemistry approaches have
shown good selectivity against point mutations18,31,42,43 we
know of no previous examples of detecting deletions. Ad-
ditionally, we show that the FACTR approach can be suc-
cessfully employed for the detection of single nucleotide
substitutions and therefore has a potentially wide ap-
plicability for detection and discrimination of mutations.
Figure 6. Detection of 16S rRNA in bacterial lysate in solu-
tion and on beads. A) Fluorescence time course of probes
targeting 16s rRNA in E.coli lysate and controls. 5 µM UL-
DNA and Thio-DNA were incubated in lysate for at 30 °C.
This was added to 200 nM QSTAR-DNA and 400 nM TPP-
DNA in buffer. λex=497 nm and λem=519 nm. B) On-bead de-
tection of target sequence. 5 µM UL-DNA and Thio-DNA and
100 nM WT template were incubated for 20 h in 70 mM Tris-
Borate buffer pH = 7.0, 10 mM MgCl2 at 30 °C. This was add-
ed to a buffered solution containing QSTAR DNA-modified
beads and 5 µM TPP-DNA and imaged without washing after
incubating for 3 h at 30 °C. C) Control bead incubated under
the same conditions with probes but without template.
Solid support. For convenient and high-throughput
analysis, many genetic detection methods rely on probes
attached to a surface.38,39 Therefore, we tested if the
FACTR nucleic acid-templated reaction network would be
suitable for detection of templates on a solid support, by
immobilizing the final signaling QSTAR probe. In princi-
ple, a target nucleic acid could catalyze the ligation reac-
tion between UL-DNA and Thio-DNA in solution, and
this ligated product could template the reaction on the
bead surface between TPP-DNA and immobilized
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Journal of the American Chemical Society
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Smith, S. J.; Nemr, C. R.; Kelley, S. O. J. Am. Chem. Soc.
2017, 139, (3), 1020.
The current approach is isothermal and non-
enzymatic and solely depends on the presence of target
sequence. The fluorogenic nature of the method ensures a
convenient readout and renders this approach suitable for
the detection of genetic sequences without sample purifi-
cation and gel electrophoresis. The possibility of using
various fluorophores potentially allows for multicolor
detection of several RNAs at the same time.44 Moreover,
since the reactions do not rely on enzyme activity and
thermocycling, they can easily be performed in a biologi-
cal context. Indeed, our results show that the coupled
nucleic-acid templated reactions perform well to detect
rRNA directly in bacterial lysate. This drastically reduces
sample handling and purification and increases accuracy,
because the target sequence can be detected directly in
lysate without the need of isolation.
The combination of FACTR with bead-supported de-
tection potentially allows for high-throughput analysis of
samples.39 The fluorescent product of the reaction is cap-
tured on the solid surface, keeping background levels low
and eliminating the need for wash steps, rendering this an
exceptionally simple method for the detection of genetic
sequences.
Future experiments will be directed at decreasing
the reaction time and lowering background signal for
even greater sensitivity. The QUAL reaction exhibits
moderate reaction rates that may be increased by using
stronger nucleophiles such as phosphorotrithioates45 or
by fine-tuning the leaving group ability. Furthermore, by
employing doubly quenched QSTAR-DNA probe
designs,35 the background signal can likely be reduced,
which is expected to increase the limit of detection of
FACTR yet further.
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ASSOCIATED CONTENT
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Further experimental details and spectra, Fig. S1-S4 and oli-
gonucleotide structures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
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*kool@stanford.edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
We are grateful for support from the U.S. National Institutes
of Health (GM068122) and The Netherlands Organisation for
Scientific Research (RUBICON) and EMBO ALTF 934-2014 to
WAV.
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