Chapter 12

Hybridization Chain Reaction for Direct mRNA Detection

Without Nucleic Acid Purification
Yao Xu and Zhi Zheng

Abstract
Hybridization chain reaction (HCR) provides a feasible solution for nucleic acid detection without target
amplification. By highly specific sandwich hybridization, target RNA can be directly captured onto solid
support and detected using HCR with fluorescent dyes. Here, we describe a novel method for malaria RNA
detection based on sandwich hybridization and two-dimensional HCR, without involving nucleic acid
purification or any enzymatic reaction, using ordinary oligonucleotides without labeling or modification.

Key words Hybridization chain reaction, mRNA detection, Sandwich hybridization

1 Introduction

Hybridization chain reaction is a new class of enzyme-free fluores-

cent signal amplification method for nucleic acid detection. In this
procedure, the target DNA/RNA initiates a hybridization cascade
between two hairpin sequences through toehold mediated strand
displacement to realize signal amplification [1, 2]. The two species
of hairpin monomers of the HCR are metastable and can coexist in
the solution until triggered by the target DNA to form a nicked
DNA double helix analogous to alternating copolymers (Fig. 1),
which can be seen in agarose gel electrophoresis (Fig. 2). Although
HCR-based signal amplifications have been described for nucleic
acid biosensing [3, 4], there are several drawbacks significantly
impeding the practical application of these methods: (1) as a highly
sensitive signal amplification method, HCR inevitably amplifies
backgrounds, decreasing specificity. These backgrounds could be
produced by physical nonspecific adsorption of hairpin sets, or
caused by leakiness in toehold-mediated HCR (i.e., hairpin poly-
merization in the absence of initiating target DNA), especially for
real sample detection when the components are complicated. (2)
The sensitivity of toehold-mediated HCR amplification is not

Imre Gaspar (ed.), RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_12, © Springer Science+Business Media LLC 2018

187
 c (Loop) a
b b

b(Stem) b b b c a

a(Toehold) c b b b b
H1 H2
Hairpin set
Triggers chain
a a c c
b a reaction
Target (initiator)
Cascade chain b b
reaction
a a
Nicked double strand ...

Fig. 1 Linear hybridization chain reaction in solution. All hairpin probes have a structure of toehold-stem-loop.
Letters marked with asterisk are complementary to the corresponding unmarked letter. Target DNA hybridizes
with hairpin in H1 via base pairing to single-stranded toehold “a”, mediating a branch migration that opens the
stem of the hairpin, exposing a “c*-b*” sticky end. This complex further opens hairpin H2 by base pairing to
“c*” to form a new complex containing a “b*-a*” sticky end, which is the same to the target. Thus, a cascade
chain reaction is generated to form a nicked double strand polymer. Figure adapted from [5] with permission
from Elsevier

Fig. 2 Effect of initiator concentration on HCR amplification. Lanes 1 and 2: 1 μM A1 and 1 μM A2,
respectively. Lane 3: DL2000 DNA marker. Lanes 4–7: four different concentrations of initiator (100, 50,
10, and 0 nM) in a 1 μM mixture of A1 and A2. Figure adapted from [5] with permission from Elsevier
 Hybridization Chain Reaction for Direct RNA Detection 189

The 1st dimension HCR The 2 nd dimension HCR

...
...
X2 ... X1 X2
X1 A1
X1
A2
X2 A2
X1 X2 ... A2 X1
X2

Target
... X1 X2
X1 X2
A1
Target A1
Hybridize and wash
Hybridize and wash

Fig. 3 Two-dimensional HCR on solid surface (see Note 10). In the 2D HCR, The target probe hybridizes on its
30 -half with the capture probe conjugated on the solid surface; the 50 -half of the target then opens the hairpin
probe X1*, triggering the cascade chain reaction of the hairpin set X* to form a nicked double helix with extra
single-strand hangout branches. After washing, hairpin set A is added; the overhang branches from X1*
opened A1 and initiated the HCR of the hairpin set A, generating a 2D HCR product. SYBR Green I is employed
in the detection phase to generate fluorescent signals. We also detected the fluorescent signal generated by
hairpin sets with the same sequences but labeled with fluorescein isothiocyanate (FITC). It was observed that
unlabeled hairpin probes generated higher signal than labeled probes [5]. Figure adapted from [5] with
permission from Elsevier

sufficient for clinical applications. Linear, one-dimensional chain

reactions are adopted in most current HCR amplification methods.
Although branched HCR or other higher dimensional amplifica-
tion may presumably give much higher sensitivity, the challenges of
steric hindrance and the difficulties to form branched oligonucleo-
tides in solution still make it hard to realize.
Here, we demonstrate an improved HCR-based RNA detec-
tion method (Figs. 3 and 4) [5]. By adopting a sandwich RNA
capturing assay, the nucleic acid extraction procedure is bypassed,
allowing high-throughput, ELISA-like sample processing in 96-
well plates. Simultaneously, a novel, onsite two-dimensional
branched HCR assembly is made possible by detecting target cap-
tured on solid support. The sensitivity and the specificity of RNA
detection were improved compared with the traditional linear HCR.

2 Materials

All oligonucleotides are purchased commercially and are PAGE-

purified. Ultrapure water and analytical grade reagents are used in
all runs. 5
SSC buffer (750 mM NaCl, 75 mM sodium citrate,
pH 7.4) is used for all hybridization reactions unless indicated
specifically.

2.1 Agarose Gel 1. Standard equipment of running agarose gels (electrophoresis

Electrophoresis tank, power supply, gel tray, and combs).
190 Yao Xu and Zhi Zheng

Target RNA

CEs Mix lysate with

all probes
LEs
Capture Stage

Capture Probe

Adaptor

Add hairpin
Set X

!
!

!
Amplification Stage

Add hairpin
Set A
...
...
...

... ... ...

... ... ...

... ... ...

Fig. 4 Schematic illustration of the direct RNA detection assay. For each target
RNA, the capture hybridization method is done with a series of oligonucleotide
probes (CEs and LEs), each of these probes containing a target-specific
sequence which is complementary to a different region of the target, and an
additional “tail” sequence that is independent of the target sequence but can
interact with either the solid support (CEs) or the adaptor (LEs). The CEs and LEs
sandwich the target RNA through target-specific hybridizations. The common CE
tail sequences hybridize to the capture probe conjugate on the surface of each
well in 96-well plate and capture the associated target RNA onto the plate. The
adaptors bind to the common LE tails and initiate the on-site 2D HCR after the
addition of hairpins. Figure adapted from [5] with permission from Elsevier
 Hybridization Chain Reaction for Direct RNA Detection 191

2. UV transilluminator.
3. Resolving gel buffer and running buffer (1
TBE): 90 mM
Tris, 89 mM boric acid, and 2.0 mM EDTA, pH 8.0.
Add about 100 mL water to a 1 L graduated cylinder. Weigh
10.89 g Tris, 5.50 g boric acid, and 0.75 g EDTA, and then
transfer to the cylinder. Add water to a volume of 900 mL. Mix
and adjust pH with HCl. Make up to 1 L with water. Store at
room temperature.
4. 1% Agarose with 1 ng/mL ethidium bromide (see Note 1).
5. DL2000 DNA marker: Store at 4  C.
6. Loading buffer.

2.2 RNA Detection 1. Tabletop centrifuge with plate adapter.

2. Heat incubator.
3. Fluorescence plate reader.
4. Tinfoil sealing film.
5. Dissolving buffer: TE.
6. Stock hybridization buffer (20
SSC): 3 M NaCl, 0.3 M
sodium citrate, store at 4  C.
7. Hybridization buffer: 5
SSC (diluted from 20
SSC), store at
4  C.
8. Wash buffer: 0.1
SSC containing 0.3 g/L lithium dodecyl
sulfate, store at 4  C.
9. Capture 96-well plate functionalized with a 22-base DNA
sequence (capture probe, Fig. 4), (Diacurate), store at 4  C
(see Note 2).
10. Blood sample with P. falciparum, store at 20  C (see Note 3).
11. Lysis mixture (Diacurate), store at room temperature.
12. 50 mg/mL Proteinase K, store at 20  C (see Note 3).
13. Hairpin sets: DNA sequences designed independently using
NUPACK [5] (http://www.nupack.org).
14. 100 μM oligonucleotide probes in TE: include CEs and LEs,
designed by Diacurate to specifically capture malaria RNA,
store at 20  C. Dilute all probes to 10 μM with hybridization
buffer before use (see Note 4).
l CE (Capture Extender).
CE probe contains two regions: region I on 30 -end has the
complementary sequence with capture probe and region II
on 50 -end can hybridize with target RNA. A “TTTTT”
sequence is inserted between the two regions.
l LE (Label Extender).
192 Yao Xu and Zhi Zheng

LE probe contains two regions: region I on 30 -end hybri-

dizes with adaptor probe and region II on 50 -end has com-
plementary sequence with target RNA. A “TTTTT”
sequence is inserted between the two regions.
l A1 50 ACA CA GAA AGG GAA ACG AGT CC CGT
TTC CCT TTC.
l A2 50 CGT TTC CCT TTC TGT GT GAA AGG GAA
ACG GGA CT.
l X1* 50 CGT TTC CCT TTC TGT GT TTTTT ATA TCC
CTC GCC GAA TCC TAG ACT CAA AGT AGT CTA
GGA TTC GGC GAG.
X1*0 consists of two regions: region I on 30 -end has the same
sequence with probe X1 and region II on 50 -end is a hangout
branch that can initiate the polymerization of hairpin set A.
A “TTTTT” sequence is inserted into the two regions.
l X2 50 AGT CTA GGA TTC GGC GAG GGA TAT CTC
GCC GAA TCC TAG ACT ACT TTG.
l Italic sequence indicates the toehold region, bold typeface
indicates loop sequences, and underline indicates stem
sequences.
15. Target oligo: 50 AGT CTA GGA TTC GGC GAG GGA TAT
TTTTT CTC TTG GAA AGA AAG TG.
The target oligo can hybridize on its 30 -half with the capture
probe on a solid support; the 50 -half of “Target oligo” can
initiate the polymerization of hairpin set X*. A “TTTTT”
sequence is inserted between the two regions.
16. Adaptor: AGT CTA GGA TTC GGC GAG GGA TAT TTTTT
ATG CTT TGA CTC AGA AAA CGG TAA CTT C.
17. 4
SYBR Green I: dilute with 4
SSC before use, store at 4  C
(see Note 5).

3 Methods

Carry out all procedures at room temperature unless otherwise

specified.

3.1 Preparation 1. All hairpin probes are heated to 95  C for 2 min and then
allowed to cool to room temperature for 1 h before use.

3.2 Linear HCR by 1. Mix 7 μL of water, 1 μL of probe A1, and 1 μL of probe A2,
1.5% Agarose Gel 1 μL of target with different concentrations and 1 μL of water
Electrophoresis for control, incubate at room temperature for 4 h.
 Hybridization Chain Reaction for Direct RNA Detection 193

2. Add loading buffer to each of the reactions, mix thoroughly

and centrifuge for several seconds.
3. Carefully load DL2000 DNA marker and samples into differ-
ent lanes of the gel.
4. Run agarose gels at 150 V for 40 min, until the sample line
reaches approximately 75–80% of the way down the gel.
5. Visualize the gels at UV light (Fig. 2).

3.3 Two- 1. Dilute target probe (Target oligo) to 10, 1, 0.1, 0.01, and
Dimensional HCR for 0.001 nM respectively in hybridization buffer (see Note 6).
an Oligo Target on 2. Add 100 μL of above solutions to individual capture wells.
Solid Surface (See
3. Seal the wells with tinfoil sealing film and incubate at 46  C for
Fig. 3) 1 h (see Note 7).
4. Tear the film, then quickly decant the wells and wash with
300 μL of wash buffer for three times, with quick decanting
in between (see Note 8).
5. Centrifuge the plate upside-down for 1 min at 600
g after the
final wash and decant.
6. Mix 120 μL of hybridization buffer, 15 μL of probe X1*, and
15 μL of probe X2 (see Notes 9 and 10).
7. Add 100 μL of above mixture to the capture plate, seal and
incubate at room temperature for 2 h.
8. Repeat wash steps (steps 4 and 5).
9. Mix 120 μL of hybridization buffer, 15 μL of probe A1, and
15 μL of probe A2.
10. Add 100 μL of above mixture to the capture plate, seal and
incubate at room temperature for 2 h.
11. Repeat wash steps (steps 4 and 5).
12. Add 150 μL of 4
SYBR Green I in 4
SSC buffer to the
reaction wells, and then incubate at room temperature for
15 min in the dark place.
13. Quantify the resulting fluorescence with a plate reader.

3.4 Direct RNA 1. Thaw the blood sample with P. falciparum at 4  C before use
Detection in Blood (see Note 3).
Sample (See Fig. 4, 2. Lyse 10 μL of thawed blood sample with 50 μL of lysis mixture,
Note 11) 85 μL of water, and 2 μL of 50 mg/mL proteinase K at 60  C
for 1 h with vigorous shaking.
3. Mix the lysate with 1.5 μL of respective CEs and LEs probes
(see Note 12).
4. Add 100 μL of the above mixture to the capture plate, then seal
the wells with tin foil and incubate at 58  C overnight without
shaking (see Note 13).
194 Yao Xu and Zhi Zheng

5. Quickly decant the wells and wash with 300 μL of wash buffer
for three times, with quick decanting in between.
6. Centrifuge the plate upside-down for 1 min at 600
g after the
final wash and decant.
7. Add 100 μL of 1 μM adaptor probe in hybridization buffer to
the wells.
8. Seal the wells with tinfoil sealing film and incubate at 46  C for
1 h.
9. Tear the film, then quickly decant the wells and wash with
300 μL of wash buffer for three times, with quick decanting
in between.
10. After the final wash, centrifuge the plate upside-down for 1 min
at 600
g to remove residual liquid.
11. Mix 120 μL of hybridization buffer, 15 μL of probe X1*, and
15 μL of probe X2 (the final concentration of each hairpin
probe is 1 μM).
12. Add 100 μL of the above mixture to the capture plate and seal
the wells, incubate at room temperature for 2 h.
13. Repeat wash steps (steps 9 and 10).
14. Mix 120 μL of hybridization buffer, 15 μL of probe A1, and
15 μL of probe A2.
15. Add 100 μL of above mixture to the capture plate, seal and
incubate at room temperature for 2 h.
16. Repeat wash steps (steps 9 and 10).
17. Add 150 μL of 4
SYBR Green I in 4
SSC buffer to the
reaction wells, and then incubate at room temperature for
15 min in the dark place.
18. Quantify the resulting fluorescence with a plate reader.

4 Notes

1. Ethidium bromide should be kept away from light in room

temperature. As EtBr is a known mutagen, be careful when
operate with this chemical and during the process of agarose gel
electrophoresis.
2. Take the plate out from refrigerator and warm to room tem-
perature before use. The capture plate is sealed with plastic film,
so tear the film of specific wells where you want to add your
sample to.
3. Blood sample and Proteinase K should be thawed at 4  C and
operated on ice throughout the procedure.
 Hybridization Chain Reaction for Direct RNA Detection 195

4. Centrifuge the lyophilized oligonucleotides at 600
g for

2 min and carefully add TE to the tube after centrifuging to
dissolve the probes to 100 μM with vigorous shaking.
5. SYBR Green I must be protected from light and stored at 4  C.
The concentration of SYBR Green I stock solution is 10,000

and the optimal final concentration is 4
. Dilute directly from
stock solution before use.
6. Target probe (Target oligo) consists of two regions that linked
with a ‘TTTTT’ structure. The first region located on the 30 -
end can hybridize with capture probe in the 96-well plate, and
the second region on the 50 -end triggers the cascade chain
reaction of two hairpin sets.
7. Make sure the wells are completely sealed so the solution in the
well won’t evaporate during incubation.
8. Unlike previously reported HCR systems in solution, our assay
is an on-site amplification technology. Leaky hairpins will be
washed off without contributing to the background. Thus, we
can maximize polymerization without being overly concerned
about leakiness by using shorter hairpins, improving the sensi-
tivity and making hairpin design easier.
9. Mix the hairpin probes just before use. Probe X1* and probe
X2 are paired with final concentration of 1 μM. Cascade chain
reaction will be triggered in the presence of target probe.
10. Hairpin probe X1* has an additional hangout sequence at the
50 -end that does not form double strand structure after poly-
merization, but instead, act as the initiating probe for the
second step of polymerization.
11. The direct RNA detection assay is comprised of two parts: the
first part is a specific RNA capture, and the second part is signal
amplification through 2-dimensional hybridization chain reac-
tions. Multiple LE probes per target could be a first step to
signal amplification by providing many initial probes for the
subsequent detection. Adaptor probes that simultaneously
hybridize to the tails of LEs mediate the capture and amplifica-
tion systems. The introduction of adaptor probe enables inde-
pendent designing of DNA probes in capture stage and
detection stage, making the assay more flexible. As a result,
the method can be used to detect any suitable RNA or DNA
targets by designing new target-specific CEs and LEs probes,
while keeping the same hairpin sets used in this study. Also, a
sequence of “TTTTT” is inserted into the two binding regions
of adaptor probe, resulting in better signal amplification than
that without “TTTTT”.
12. Multiple oligonucleotide probes capture the RNA on 96-well
plate in the capture stage. Based on our previous study, this
196 Yao Xu and Zhi Zheng

design offered cooperative hybridization between the capture

probes on the solid surface and the multiple CEs binding to
one target, presumably leading to a stronger capture than
single CE–capture probe interaction [5], which makes sure
that only the specific target can be captured to the solid surface
and further trigger downstream cascade reactions.
13. As the hybridization is taken place at a temperature (58  C)
that is higher than the annealing temperature of single CE or
LE probe, single probes or hairpins will be washed off and
cannot be stably immobilized on the solid surface to cause
nonspecific absorption. Leaky hairpin polymerization products
without binding to the target will be washed off as well without
causing background.

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