- Email: [email protected]
Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancer-related gene
Accepted Manuscript Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancer-related gene Jianguo Xu, Zai-Sheng Wu, Zhenmeng Wang, Hongling Li, Jingqing Le, Lee Jia PII:
S0142-9612(16)30193-4
DOI:
10.1016/j.biomaterials.2016.05.020
Reference:
JBMT 17502
To appear in:
Biomaterials
Received Date: 25 January 2016 Revised Date:
20 April 2016
Accepted Date: 17 May 2016
Please cite this article as: Xu J, Wu Z-S, Wang Z, Li H, Le J, Jia L, Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancer-related gene, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Two-wheel drive-based DNA nanomachine and its sensing potential for highly
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sensitive analysis of cancer-related gene
Jianguo Xu, Zai-Sheng Wu*, Zhenmeng Wang, Hongling Li, Jingqing Le, and Lee Jia*
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Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry; Fujian Provincial Key Laboratory of Cancer
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Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350002, China.
*Correspondence should be addressed to Lee Jia and Zai-Sheng Wu, CMAPC, Fuzhou University, Fuzhou, Fujian 350002, China. Phone: 086-591-8357-6912. Email: [email protected] (L. Jia) and [email protected] (Z.S.
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Wu).
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Abstract With the biological significance and important advances of nano-scale DNA devices, scientific
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activities have been directed toward developing molecular machinery. In this work, we present a novel two-wheel drive-based DNA nanomachine composed of one signaling recognition probe (SRP), one label-free recognition probe (LRP), and one driving primer (DP). Target DNA hybridization can
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activate LRP-based wheel driving by resorting to DP-mediated polymerization/nicking/displacement cycles. This in turn results in the accumulation of nicked strand 1 (NS1) that can initiate extended
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SRP-based wheel driving. As a result, the hairpin structure of SRP is stretched and pre-quenched fluorescence is restored. Meanwhile, lots of nicked strand 2 (NS2) are produced, which could hybridize perfectly with SRP and lead to further fluorescence amplification. It is worth noting that, because the nanomachine operation relies strongly on inputted target trigger, the unwanted
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background is completely eliminated. The detection limit of 1 pM and an excellent capability to recognize the single-base mutation were achieved. Significantly, the interrogating of target trigger extracted from cancer cells is already available, reflecting the potential for practical applications. As
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a proof-of-concept building, the unique analytical properties would significantly benefit the DNA
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nanomachines and reveal great promise in biochemical and biomedical studies.
Keywords: two-wheel drive; DNA nanomachine; signal amplification; cancer-related gene
1. Introduction The exquisite sequence predictability, biocompatibility, interaction specificity, and diversity of DNA strands make DNAs useful building materials for engineering devices at the nanoscale [1-5]. This
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has enabled the construction of molecular machinery from DNA oligonucleotides that rely on the programmed sequence-specific interaction, and a diverse and rapidly growing set of DNA
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nanomachines have been extensively exploited [6-9]. In fact, DNA nanomachines are artificially designed assemblies that switch between defined conformations in response to an external chemical or biological stimuli autonomously, which was capable of performing specific functions, such as
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biosensing, diagnostics, and targeted therapies [6, 10-13]. Within the past few decades, the emerging research fields of isothermal amplification, including strand displacement amplification (SDA)
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[14-16], rolling circle amplification (RCA) [17-19], and hybridization chain reaction (HCR) [20-22], are able to translate various input stimuli into certain signals. This provides particularly exciting possibilities for the advanced development of smart DNA nanomachines [11, 23]. Obviously, it is a surprising extension of isothermal amplification to convert static DNA structures into machines,
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representing a subset of available devices and ongoing research.
Gene disorder is a complex phenomenon that varies widely in the genetic diseases [24]. Especially in the roots and progression of cancers [25], many different genes have been found to be
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predominantly encoded, and the defective molecules within the malignant cells are major causes, as well as the potential targets for diagnosis and therapy. Much endeavor has thus been paid to the gene
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analysis field. Given the distinctive properties of precise control, versatile design and synthesis, fast response, and usual activation by nucleic acids, some types of DNA nanomachines have been reported to give the efficient nucleic acid detection with different levels of success. For instance, the group of Itamar Willner reported an autonomous DNA-based machine that amplifies the detection of M13 phage single-stranded with the sensitivity that rivals the polymerization chain reaction (PCR) [26]. Zhang et al. created a new type of nanomachine that harnesses specific target binding to trigger
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the assembly of separate DNA components [27]. Despite good performances, these DNA nanomachines were still confronted with some limitations with respect to delicate protocols, complex
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operation, and insufficient sensitivity and specificity [13, 28]. Therefore, the demand remains high for developing powerful DNA nanomachines with improved assay performance.
In this work, by using cancer-related gene, p53 [29], as the fueled molecule, a two-wheel
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drive-based DNA nanomachine was explored consisting of two types of recognition probes, termed as signaling recognition probe (SRP) and label-free recognition probe (LRP), and one driving primer
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(DP). SRP and LRP were prepared by functionalizing the oligonucleotides with a half recognition site of nickase in the middle region and a region at the swing arms partly complementary to target DNA. The target trigger acts as a bridge to align the two probes together, facilitating subsequent hybridization
between
LRP
and
DP.
On
this
basis,
LRP-based
wheel
driving
by
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polymerization/nicking is processed, leading to substantially accumulation of nicked strand 1 (NS1) through strand displacement. Meanwhile, the 3’ end extension of SRP over target trigger was proceed, in which the produced NS1 could compete with the target trigger to hybridize with the extended part.
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And then the second wheel driving was autonomously induced in a similar manner. During the period, the fluorescence of SRP was amplified. It’s worthy to mention that the resultant product of
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nicked strand 2 (NS2) could directly hybridize to SRP, giving rise to further fluorescent emission remarkably. Consequently, the two-wheel drive-based DNA nanomachine was successfully constructed and worked cooperatively to give a signal amplification. One significant advantage that should be mentioned was that the nanomachine operation was absolutely depend on the inputted trigger, bypassing the undesired background entirely when used for sensing. Therefore, the intelligent proposed signal amplification via combining with stringently suppressed background
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enabled the detection of as low as 1 pM target trigger accompanied with a dynamic measurable range from 1 pM to 8 nM. Moreover, the single-base mutation could be easily discriminated against
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perfectly complementary target trigger especially when the mutation point was located at the junction site. Importantly, the target trigger generated from genomic DNA based asymmetric polymerization chain reaction can induce considerable signal readout, suggesting great promise for practical
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applications. The conceptually designed two-wheel drive-based DNA nanomachine as well as distinct assay ability would not only promote the development of more attractive DNA nanodevices,
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but also create the new opportunity for a wide range of biological purposes.
2. Experimental section 2.1 Materials and reagents
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The oligonucleotide of SRP (HPLC purified) was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China), and other strands (PAGE purified) were obtained from Invitrogen Bio Inc. (Shanghai, China). Their sequences are listed in Table 1, and
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the secondary structures of all oligonucleotides were predicted by online bioinformatic software (http://mfold.ma.albany.edu/). Klenow fragment (3’→ 5’ exo-) polymerase, Nt. BbvCI nicking
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endonuclease, and low DNA ladder were bought from New England Biolabs (USA) Ltd. Deoxynucleotide triphosphates (designated as dNTPs) and Sybr Green I were purchased from Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China), while the universal genomic DNA exaction kit was from TaKaRa Biotechnology Co. Ltd. (Dalian, China). The 25 mM tris-buffer (PH= 8.2) containing 100 mM NaCl, 50 mM KAc, 10 mM Mg(Ac)2, and 1 mM DTT, was used as the reaction solution in all runs.
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All other chemicals were of analytical grade and used as received. Aqueous solutions were prepared with double-distilled water processed with a Kerton lab MINI water purification system
Table 1. Oligonucleotide sequences designed in the present study.
before usage.
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(UK) with electrical resistivity of 18.25 MΩ and stored in darkness at 4
Sequence (5’-3’)
Signaling recognition probe (SRP)
(DABCYL)-agtgtggAGAATGACAATCGCAAAAAccacact(FAM) GCTGAGGAATTACAGGCACAAACA
Label-free recognition probe (LRP)
CGCACCTcaaaggtcgGCTGAGGTACAGAGTGACAcgacctttgTT TT
Driving primer 1 (DP1)
TTTTTTTTcaaaggtcg
Driving primer 2 (DP2)
TTTTTTTTTTaaggtcg
Nicked strand 1 (NS1) Nicked strand 2 (NS2)
TCAGCCGACCTTTGAGGTGCG
Target DNA
GCTTTGAGGTGCGTGTTTGTGCCTGTCCTG
Mutant target DNA 1 (MT1)
GCTTTGAGGTGCGAGTTTGTGCCTGTCCTG
Mutant target DNA 2 (MT2)
GCTTTGAGGTGCATGTTTGTGCCTGTCCTG
Mutant target DNA 3 (MT3)
GCTTTGAGATGCGTGTTTGTGCCTGTCCTG
Mutant target DNA 4 (MT4)
GCTTTGAGGTGCGTGTTTATGCCTGTCCTG
Non-target DNA (NT)
GCTTACTGGTGAACGTTTGTGATTGTCGAG
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TCAGCAGTGTGGTTTTTGCGATTGTCATTCTCCACACT
GCTTTGAGGTGCGTGTTT
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Sense primer 1
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Note
GTGAGGCTCCCCTTTCTT
Sense primer 2 Anti-sense primer 2
GAGGTAAGCAAGCAGGACA GCAAGGAAAGGTGATAAAAGT
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Anti-sense primer 1
Letters shown in lowercase in SRP and LRP indicate the self-complementary stem of hairpin structure, while the fragments in bold are the half recognition site of Nt. BbvCI. The underlined portions denote the complementary fragments of target DNA. Notably, SRP was chemically tagged with a fluorophore of FAM and a quencher of DABCYL. The boxed segments in DPs, including DP1 and DP2, are complementary to the boxed one of LRP. NS1 and NS2 are the strands nicked and displaced from LRP and extended SRP respectively (see Scheme 1). MT1, MT2, MT3, and MT4 have the same base sequence as target DNA except for the point mutation in grey background, while
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NT was the target analogue randomly designed. When performing asymmetric PCR amplifications, sense primer 1/anti-sense primer 1 and sense primer 2/anti-sense primer 2 were used for p53
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amplicons and control amplicons, respectively.
2.2 Instruments
Fluorescence measurements were carried out at room temperature on a Hitachi F-7000
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fluorescence spectrometer (Hitachi, Ltd., Japan) with a Xenon lamp as the excitation source. The samples were excited at 492 nm, and the emission spectrum collection range was 500− 600 nm.
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Fluorescence intensity at 518 nm was used to evaluate the performance of the proposed strategy. The excitation and emission slits used in the experiments were both 5 nm and the photo- multiplier tube voltage was 600 V.
A 12% native polyacrylamide gel (native-PAGE) was freshly prepared and carried out on a gel
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electrophoresis instrument (BIO-RAD, USA) at a constant voltage of 80 V for 90 min by using 0.5× TBE buffer (4.5 mM Tris, 4.5 mM boric acid, 0.1 mM EDTA, pH 7.9) as the working buffer. The resulting gel was excited and imaged using the ChemiDoc XRS system with Image Lab analysis
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software (Bio- Rad). Considering SRP was FAM modified, sybr green I was only used in samples
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where SRP were not present, for example, DNA markers. 2.3 Two-wheel drive-based DNA nanomachine for target stimulus assay The assay experiment was carried out in 25-µL solution containing 15 µL of tris-buffer, 2 µL of SRP (5 µM), 2 µL of LRP (5 µM), 2µL of DP1 (5 µM), and 2 µL of target trigger (varying concentrations). Followed by incubation at 90
for 5 min and allowed to cool down to room
temperature. Then, 0.5 µL of Klenow fragment (3’→ 5’ exo-) polymerase, 0.5 µL of Nt. BbvCI nicking endonuclease, and 1 µL of 10 mM dNTPs were added into the above solution and incubated
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for 3.5 h at 37 . Finally, the reaction was inactivated by heating at 80 °C for 20 min, and the resulting mixture was used for the fluorescence measurement after adding 175 µL of dd-H2O.
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2.4 Genomic DNA extraction and asymmetric PCR amplifications Genomic DNA extraction from cultured human lung cancer cell line A549 was performed according to universal Genomic DNA exaction kit. Asymmetric PCR amplifications were carried out
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in terms of the process reported previously [30]. Briefly, 50 µL of 1× PCR buffer containing 1 µg of genomic DNA, 0.5 µM sense primer, 0.1 µM anti-sense primer, 0.15 mM dNTP mixture, as well as
denaturation step at 94
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2.5 U taq polymerase, was stirred thoroughly, and then moved to a PCR instrument. After a 5 min , the amplification was achieved by thermal cycling for 30 cycles at 94 °C
for 30 s, 55 °C for 30 s, 72 °C for 55 s, and a final extension at 72 °C for 7 min. The PCR products until required for gel electrophoresis and fluorescent assay.
3. Results and discussion
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were stored at 4
3.1 Design and working principle of the two-wheel drive-based DNA machine
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As shown in Scheme 1, two types of RP were designed and both engineered with two functional domains. One is a recognition region that is complementary to the target gene, which was at the 3’
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end of SRP or 5’ end of LRP. The other one is a half recognition site of restriction endonuclease Nt. BbvCI (shown in yellow), which was used to initiate the amplification machine. Besides, a DP was projected with several bases complementary to the 3’ end of LRP. Note that the SRP was tagged with a pair of fluorophore and quencher (FAM and DABCYL), in which the two molecules were in close proximity by the hairpin structure and the fluorescence of FAM was quenched in the initial state. The target DNA serving as a trigger acts as a template to align a pair of SRP and LRP perfectly along it,
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and the hairpin structure of LRP was deteriorated, thereby enabling the hybridization of LRP and DP (step
). With the aid of polymerase, the extension of 3’ end of SRP over target trigger and DP over
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LRP were implemented, during which the recognition site of Nt. BbvCI was formed in the latter one, causing nickase to specifically cleave the upper strand. As a result, a number of NS1 were generated by autonomously-performed nicking/polymerization/displacement cycles, and it seems like the ). Interestingly, the produced NS1 was
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operation was in a one-wheel drive manner (step
complementary to the extended part in SRP, so that it was able to compete with target DNA to
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hybridize with extended SRP. Although the binding force of NS1 to extended SRP was not powerful enough compared with target DNA, the driving force of polymerase would promote the hybridization event between NS1 and extended SRP, and lead to the replication of extended SRP (step
). The
distance between fluorophore and quencher was thus increased due to the hairpin structure opening,
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generating the fluorescence signal. Moreover, similarly, the recognition site of Nt. BbvCI was obtained in the newly formed duplex, and a large accumulation of NS2 was achieved (step
),
which was capable of hybridizing with SRP directly and made the fluorescence of FAM greatly ). This could lead to the second wheel driving. In addition, the target DNA released
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restore (step
was reused into next cycle (step
). We can see that the LRP-based wheel driving operation yields
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the primer to activate the extended SRP-based wheel driving like molecular nanomachine. In this sense, the two-wheel drive-based DNA nanomachine was successfully established, and two types of drives worked synergistically to sense target stimuli. Specially, since target DNA acts as the trigger species to induce the conformation change of LRP and sever as polymerization template for SRP (seen step
and
), the DNA nanomachine was not allowed to run completely when target
DNA was absent, indicating that no background was detected when used to screen target molecule.
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Scheme 1. Sketch of two-wheel drive based DNA nanomachine for target stimulus sensing. Binding to target stimulus;
LRP-based wheel drive and extension of SRP over target stimulus;
Binding of NS1 to extended SRP through competition with target stimulus; Hybridization of SRP with NS2.
Target stimulus recycling to initiate the
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based wheel drive;
Extended SRP
next round. The yellow segments inserted into oligonucleotide probes indicate the cleavage site of Nt.
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BbvCI.
3.2 Mechanism demonstration by gel electrophoresis
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To confirm that two-wheel drive based DNA nanomachine can operate as expected, gel electrophoresis was performed to give a straightforward characterization under optimized conditions (see Figure S1). As displayed in Figure 1, one single and obscure band corresponding to the designed SRP was observed in lane a, suggesting the fluorescence of FAM was quenched by DABCYL. Identical band was obtained in lane b where SRP, LRP, DP1 and polymerase coexisted, while two products with low mobility were observed in lane c upon addition of target trigger. Presumably, the upper band was the ternary complex of SRP-p53-LRP, and the lower band was the extended SRP/p53
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gene duplex. In contrast, two different bright bands with obvious shift in the positions in the presence of nickase were seen in lane e, and the band of SRP was totally disappeared. The mobility change
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and consumption of SRP represent that the nickase was worked, where the upper band in this lane was belong to the replicate helix of extended SRP, and the dominantly existed lower band was the hybridized complex of SRP and NS2 (see lane g). Notably, the relevant control sample in lane d was
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still the same as lane a, indicating that the DNA nanomachine was not operated in the absence of target trigger. So no any other background bands could be found. In addition, the NS1 mediated
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polymerization/nicking/displacement cycles on extended SRP was verified in lane f. In view of that the bands were identical to those in lane e, this phenomenon clearly exhibits that the extended SRP-based wheel-drive can be activated by the product of NS1 originating from the LRP-based
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wheel driving, strongly convincing the reasonability of proposed DNA nanomachine.
Figure 1. 12% native PAGE image of different mixtures. (a) SRP; (b) SRP+LRP+DP1+Polymerase; (c)
SRP+LRP+DP1+p53+Polymerase;
(d)
SRP+LRP+DP1+Polymerase+Nickase;
(e)
SRP+LRP+DP1+p53+Polymerase+Nickase; (f) SRP+p53+NS1+Polymerase+Nickase; and (g) SRP+NS2. “M” denotes the marker.
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3.3 Feasibility investigation for stimulus sensing In order to determine that the nanomachine based signal amplification can be utilized for stimulus
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sensing, different samples were firstly prepared and the corresponding fluorescent spectra were recorded as shown in Figure 2 to clarify this point. One can see that the signal of the DNA nanomachine (curve b) in the absence of target trigger was much closed to the inherent fluorescence
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of SRP (curve a), suggesting the negligible background fluorescence. Excitingly, a significantly increased fluorescence (about 800 %) was detected when encountering with the target trigger (curve
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c), indicating it possess powerful capability for target analyte analysis. As a control test, the mixture in the absence of nickase (curve d) or polymerase (curve e) compared with line c was also performed. As anticipated, these samples could not give any detectable fluorescence increase and closely approaches to the background. The data yielded by this study provides confirmatory evidence that
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the DNA nanomachine is available for target sensing.
Figure 2. Typical fluorescent emission spectra collected from the solutions containing SRP (a), SRP+LRP+DP1+Polymerase+Nickase
(b),
SRP+LRP+DP1+p53+Polymerase+Nickase
(c),
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SRP+LRP+DP1+p53+Polymerase (d), SRP+LRP+DP1+p53+Nickase (e). The signal increase was deduced from [(F-F0)/F0], where F and F0 are the data in the presence and absence of target trigger, respectively. The concentrations of SRP, LRP, and DP1 were 500 nM, while the involved target was
3.4 Interrogating capability for target stimulus
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100 nM.
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Once possessing the ability to response to certain trigger, the sensing capability of two-wheel drive-based DNA nanomachine was verified. Figure 3A depicts the spectral data collected for various
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target concentrations ranging from 0 to 15 nM. We can see that the maximal value was obtained at 8 nM, and further increasing the target concentration would not give higher signal. Since the target DNA at 1 pM can induce a detectable signal (see inset), we define it as the detection limit. Figure 3B is the calibration curve deduced from the peak values acquired at 518 nm, and the regression
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equation is Y= 387.36+ 292.41X (R2= 0.9949) associated with a dynamic range from 1 pM to 8 nM, where Y and X represent the peak value and target trigger concentration, respectively. Undoubtedly, the excellent performance was attributed to uniquely designed signal amplification and greatly
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reduced background. In contrast to reported approaches that relying on fluorescence as output signal
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[31-36], the assay ability was apparently improved regarding to the detection limit.
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Figure 3. (A) Fluorescent spectra upon addition of various concentrations of target stimulus. The curves from bottom to top correspond to the target DNA with concentrations of 0, 1 pM, 10 pM, 100 pM, 400 pM, 700 pM, 1 nM, 2 nM, 3 nM, 6 nM, 8 nM, 10 nM and 15 nM, respectively. Inset
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amplified the curves at low target concentrations range. (B) Corresponding calibration plot obtained from the peak values at 518 nm over the range of 1 pM to 8 nM. Error bars are the standard deviations of three parallel experiments.
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Essentially, the proposed DNA nanomachine works on the basis of isothermal nucleic acid amplification. This amplification technique is a simple process that rapidly and efficiently
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accumulates nucleic acid sequences at constant temperature. Since the early 1990s, a large number of isothermal amplification systems have been described as alternatives to polymerase chain reaction (PCR). A recent pioneered review literature [37] has summarized the development of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration,
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encompassing the previous studies presented in the past two decades. An inspiring fact is that, in terms of the limit of detection, linear response range and the ability to discriminate single base mismatch, the present DNA nanomachine is superior to considerable number of fluorescent assay
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systems based on the isothermal nucleic acid amplification (see the details in Table S1).
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Despite the impressive progress made in the development of fluorescent sensing systems, however, when compared with some other transducing techniques (e.g., colorimetric assay [11, 38] and electrochemical measurements [39, 40]), the assay sensitivity is not powerful enough and still needs to be further improved to meet the demand for ultra-sensitive detection of nucleic acids. According to our own research experiences [38] and literature reports [41-43], several strategies could be employed to improve further the assay sensitivity, for example, the optimization of the base sequence of SRP and LRP [41-42] and adjustment the concentrations of oligonucleotide probes [43]. We think
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that increasing the proportion of “C-G” pairs in the stem of SRP would make FAM and DABCYL closer to each other, leading to the lower background fluorescence. In addition, since the background
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fluorescence was smart circumvented except for the inherent fluorescence of SRP, increasing the ratio of SRP/LRP would enhance further the output signal when profiling certain concentration of target stimulus. The optimizing experiments are expected to be capable of improving further the
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two-wheel drive-based DNA nanomachine sensitivity. 3.5 Stimuli specificity study
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To our knowledge, a majority of polymorphisms in the human genome are caused by mutations that involve one or more nucleotides, and the sequence-specificity detection is of great importance [44]. To evaluate whether single nucleotide mutation could be correctly distinguished from the right one with the present DNA nanomachine, four synthetic target molecules, each with a single
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nucleotide mutation in different positions were used for specificity study at the same concentration. Notably, the mutation points in MT1 and MT2 were designed at the junction site, while MT3 and MT4 were arbitrarily projected. As revealed in Figure 4, the target stimulus (wild-type p53 gene)
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could be easily differentiated from other analogues no matter where the mutation point was located, permitting the facile distinction of known sequence variants differing by only one single nucleotide.
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Importantly, the responses induced by the single nucleotide mutation at the junction sites were relatively weak, indicating the impressive progress in the detection of single nucleotide mutation. Although the mutant target molecules were likely to capture one pair of SHP and LHP, they are unable to drive efficiently the machine operate owing to the decreased matching ability. Especially when the mutant nucleotides were at the junction sites, the mismatched single nucleotide is incapable of leading to the extension of SRP over MT1 or prohibits the polymerization of NS1 over extended
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SRP that is obtained from MT2 as polymerization template. Additionally, we found that the randomly planned non-target trigger only induces a negligible fluorescence increase.
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Actually, DNA nanomachines are required to be specifically initiated by the stimulus [23]. However, it’s still a biggest challenge to detect specifically nucleic acids that differ only in a single nucleotide [45], and genetic diagnosis was currently limited by the cumbersome nature of some DNA
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sensing methods. For instance, the capacity of conventional molecular beacon is relative poor in the detection of single nucleotide substitution [46-48], while the ligation-based strategies utilizing RCA
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for mutation analysis often involved time-consuming operations, such as denaturing PAGE purification, despite of the desired specificity by high-fidelity DNA ligase [41, 49]. In contrast, the proposed DNA nanomachine was easily constructed and the corresponding specificity at the junction site was comparable to ligation-based strategies [49-51] but higher than many literature values [23,
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46, 48, 52, 53].
Figure 4. Assay specificity of the DNA nanomachine toward 100 nM target stimuli. MT1, MT2, MT3, and MT4 are single-nucleotide mutations, and NT is randomly designed oligonucleotide strand with same length as target stimulus. The relative signal response was calculated as [(F-F0)/ (Ft-F0)],
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where F0, Ft, and F are the values corresponding to samples of blank, p53 target, and MTs (MT1, MT2, MT3, and MT4) or NT, respectively. The data are presented as means ± standard deviation.
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3.6 Real samples detection Essentially, all the advancements of technology should be directed toward the practical applications, and strategies holding certain perspective in real biological samples are highly
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appreciated. Therefore, the aim of this section was to study whether the proposed DNA nanomachine has the practical potential for analyzing target molecule in genomic DNA. In light of that the DNA
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extracted from cancer cells was double-stranded, asymmetric PCR amplifications were performed first to produce corresponding single-stranded DNAs, and the results of gel electrophoreses are shown in Figure 5A. One can see that a detectable band corresponding to p53 amplicons and control amplicons was observed in lane a and lane b, respectively, indicating the successful amplifications.
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The produced single-stranded target stimulus and control stimulus could not be seen since the poor embedding ability of sybr Green I toward single-stranded DNA. Figure 5B are the spectra recorded from the asymmetric PCR amplification products. Compared with the control amplicons in which the
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value (curve b) is closed to the blank (curve a), a remarkable signal response was obtained by target
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amplicons (curve c), offering robust capability to monitor the presence of particular sequence from genomic DNA. Inset displays the specific fluorescent difference after deducting the background value, confirming further that the DNA nanomachine is adoptable for genomic DNA detection. These measured data imply practical potential for clinical relevant applications.
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Figure 5. (A) Gel electrophoresis analysis of asymmetric PCR amplification products. Lane a, p53
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amplicons; lane b, control amplicons. (B) Fluorescent spectra of two-wheel drive based DNA nanomachine for blank sample (a), control amplicons (b) and p53 target amplicons (c). Inset: comparative data on the signal triggered by different amplicons. F and F0 are the peak values in the presence and absence of amplicons, respectively.
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4. Conclusion
In aggregate, a two-wheel drive-based DNA nanomachine was herein proposed as a proof-of-concept to develop an impressive DNA nanodevice for the detection of cancer-related gene.
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This DNA molecular machine possesses three highlighted characteristics: (1) The two-wheel
and
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drive-based DNA nanomachine induce two consecutive stages of fluorescence amplification (step ), and target stimulus was recycled (step
), achieving a substantially amplified readout
signal; (2) This nanomachine uses target DNA as fuel to cause LRP-based wheel drive to communicate with extended SRP based drive, while the usually brought background in the absence of stimulus was entirely circumvented; (3) The protocol is highly specific due to the stringently designed amplification pathway, which allows easy discrimination of sequences with one nucleotide mutation. In such a situation, the two-wheel-drive-based DNA nanomachine was a progressive
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achievement in material science, molecular computing, and biosensing, offering applicable routes
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toward achieving versatile and powerful molecular machines.
Acknowledgments
This work was supported by Ministry of Science and Technology of China (2015CB931804),
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National Natural Science Foundation of China (NSFC) (grant# 81273548, 21275002, and U1505225), and Independent Research Project of State Key Laboratory of Photocatalysis on Energy
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and Environment (NO. 2014CO1).
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S0142-9612(16)30193-4
DOI:
10.1016/j.biomaterials.2016.05.020
Reference:
JBMT 17502
To appear in:
Biomaterials
Received Date: 25 January 2016 Revised Date:
20 April 2016
Accepted Date: 17 May 2016
Please cite this article as: Xu J, Wu Z-S, Wang Z, Li H, Le J, Jia L, Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancer-related gene, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Two-wheel drive-based DNA nanomachine and its sensing potential for highly
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sensitive analysis of cancer-related gene
Jianguo Xu, Zai-Sheng Wu*, Zhenmeng Wang, Hongling Li, Jingqing Le, and Lee Jia*
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Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry; Fujian Provincial Key Laboratory of Cancer
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Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350002, China.
*Correspondence should be addressed to Lee Jia and Zai-Sheng Wu, CMAPC, Fuzhou University, Fuzhou, Fujian 350002, China. Phone: 086-591-8357-6912. Email: [email protected] (L. Jia) and [email protected] (Z.S.
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Wu).
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Abstract With the biological significance and important advances of nano-scale DNA devices, scientific
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activities have been directed toward developing molecular machinery. In this work, we present a novel two-wheel drive-based DNA nanomachine composed of one signaling recognition probe (SRP), one label-free recognition probe (LRP), and one driving primer (DP). Target DNA hybridization can
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activate LRP-based wheel driving by resorting to DP-mediated polymerization/nicking/displacement cycles. This in turn results in the accumulation of nicked strand 1 (NS1) that can initiate extended
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SRP-based wheel driving. As a result, the hairpin structure of SRP is stretched and pre-quenched fluorescence is restored. Meanwhile, lots of nicked strand 2 (NS2) are produced, which could hybridize perfectly with SRP and lead to further fluorescence amplification. It is worth noting that, because the nanomachine operation relies strongly on inputted target trigger, the unwanted
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background is completely eliminated. The detection limit of 1 pM and an excellent capability to recognize the single-base mutation were achieved. Significantly, the interrogating of target trigger extracted from cancer cells is already available, reflecting the potential for practical applications. As
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a proof-of-concept building, the unique analytical properties would significantly benefit the DNA
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nanomachines and reveal great promise in biochemical and biomedical studies.
Keywords: two-wheel drive; DNA nanomachine; signal amplification; cancer-related gene
1. Introduction The exquisite sequence predictability, biocompatibility, interaction specificity, and diversity of DNA strands make DNAs useful building materials for engineering devices at the nanoscale [1-5]. This
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has enabled the construction of molecular machinery from DNA oligonucleotides that rely on the programmed sequence-specific interaction, and a diverse and rapidly growing set of DNA
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nanomachines have been extensively exploited [6-9]. In fact, DNA nanomachines are artificially designed assemblies that switch between defined conformations in response to an external chemical or biological stimuli autonomously, which was capable of performing specific functions, such as
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biosensing, diagnostics, and targeted therapies [6, 10-13]. Within the past few decades, the emerging research fields of isothermal amplification, including strand displacement amplification (SDA)
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[14-16], rolling circle amplification (RCA) [17-19], and hybridization chain reaction (HCR) [20-22], are able to translate various input stimuli into certain signals. This provides particularly exciting possibilities for the advanced development of smart DNA nanomachines [11, 23]. Obviously, it is a surprising extension of isothermal amplification to convert static DNA structures into machines,
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representing a subset of available devices and ongoing research.
Gene disorder is a complex phenomenon that varies widely in the genetic diseases [24]. Especially in the roots and progression of cancers [25], many different genes have been found to be
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predominantly encoded, and the defective molecules within the malignant cells are major causes, as well as the potential targets for diagnosis and therapy. Much endeavor has thus been paid to the gene
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analysis field. Given the distinctive properties of precise control, versatile design and synthesis, fast response, and usual activation by nucleic acids, some types of DNA nanomachines have been reported to give the efficient nucleic acid detection with different levels of success. For instance, the group of Itamar Willner reported an autonomous DNA-based machine that amplifies the detection of M13 phage single-stranded with the sensitivity that rivals the polymerization chain reaction (PCR) [26]. Zhang et al. created a new type of nanomachine that harnesses specific target binding to trigger
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the assembly of separate DNA components [27]. Despite good performances, these DNA nanomachines were still confronted with some limitations with respect to delicate protocols, complex
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operation, and insufficient sensitivity and specificity [13, 28]. Therefore, the demand remains high for developing powerful DNA nanomachines with improved assay performance.
In this work, by using cancer-related gene, p53 [29], as the fueled molecule, a two-wheel
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drive-based DNA nanomachine was explored consisting of two types of recognition probes, termed as signaling recognition probe (SRP) and label-free recognition probe (LRP), and one driving primer
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(DP). SRP and LRP were prepared by functionalizing the oligonucleotides with a half recognition site of nickase in the middle region and a region at the swing arms partly complementary to target DNA. The target trigger acts as a bridge to align the two probes together, facilitating subsequent hybridization
between
LRP
and
DP.
On
this
basis,
LRP-based
wheel
driving
by
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polymerization/nicking is processed, leading to substantially accumulation of nicked strand 1 (NS1) through strand displacement. Meanwhile, the 3’ end extension of SRP over target trigger was proceed, in which the produced NS1 could compete with the target trigger to hybridize with the extended part.
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And then the second wheel driving was autonomously induced in a similar manner. During the period, the fluorescence of SRP was amplified. It’s worthy to mention that the resultant product of
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nicked strand 2 (NS2) could directly hybridize to SRP, giving rise to further fluorescent emission remarkably. Consequently, the two-wheel drive-based DNA nanomachine was successfully constructed and worked cooperatively to give a signal amplification. One significant advantage that should be mentioned was that the nanomachine operation was absolutely depend on the inputted trigger, bypassing the undesired background entirely when used for sensing. Therefore, the intelligent proposed signal amplification via combining with stringently suppressed background
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enabled the detection of as low as 1 pM target trigger accompanied with a dynamic measurable range from 1 pM to 8 nM. Moreover, the single-base mutation could be easily discriminated against
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perfectly complementary target trigger especially when the mutation point was located at the junction site. Importantly, the target trigger generated from genomic DNA based asymmetric polymerization chain reaction can induce considerable signal readout, suggesting great promise for practical
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applications. The conceptually designed two-wheel drive-based DNA nanomachine as well as distinct assay ability would not only promote the development of more attractive DNA nanodevices,
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but also create the new opportunity for a wide range of biological purposes.
2. Experimental section 2.1 Materials and reagents
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The oligonucleotide of SRP (HPLC purified) was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China), and other strands (PAGE purified) were obtained from Invitrogen Bio Inc. (Shanghai, China). Their sequences are listed in Table 1, and
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the secondary structures of all oligonucleotides were predicted by online bioinformatic software (http://mfold.ma.albany.edu/). Klenow fragment (3’→ 5’ exo-) polymerase, Nt. BbvCI nicking
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endonuclease, and low DNA ladder were bought from New England Biolabs (USA) Ltd. Deoxynucleotide triphosphates (designated as dNTPs) and Sybr Green I were purchased from Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China), while the universal genomic DNA exaction kit was from TaKaRa Biotechnology Co. Ltd. (Dalian, China). The 25 mM tris-buffer (PH= 8.2) containing 100 mM NaCl, 50 mM KAc, 10 mM Mg(Ac)2, and 1 mM DTT, was used as the reaction solution in all runs.
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All other chemicals were of analytical grade and used as received. Aqueous solutions were prepared with double-distilled water processed with a Kerton lab MINI water purification system
Table 1. Oligonucleotide sequences designed in the present study.
before usage.
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(UK) with electrical resistivity of 18.25 MΩ and stored in darkness at 4
Sequence (5’-3’)
Signaling recognition probe (SRP)
(DABCYL)-agtgtggAGAATGACAATCGCAAAAAccacact(FAM) GCTGAGGAATTACAGGCACAAACA
Label-free recognition probe (LRP)
CGCACCTcaaaggtcgGCTGAGGTACAGAGTGACAcgacctttgTT TT
Driving primer 1 (DP1)
TTTTTTTTcaaaggtcg
Driving primer 2 (DP2)
TTTTTTTTTTaaggtcg
Nicked strand 1 (NS1) Nicked strand 2 (NS2)
TCAGCCGACCTTTGAGGTGCG
Target DNA
GCTTTGAGGTGCGTGTTTGTGCCTGTCCTG
Mutant target DNA 1 (MT1)
GCTTTGAGGTGCGAGTTTGTGCCTGTCCTG
Mutant target DNA 2 (MT2)
GCTTTGAGGTGCATGTTTGTGCCTGTCCTG
Mutant target DNA 3 (MT3)
GCTTTGAGATGCGTGTTTGTGCCTGTCCTG
Mutant target DNA 4 (MT4)
GCTTTGAGGTGCGTGTTTATGCCTGTCCTG
Non-target DNA (NT)
GCTTACTGGTGAACGTTTGTGATTGTCGAG
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TCAGCAGTGTGGTTTTTGCGATTGTCATTCTCCACACT
GCTTTGAGGTGCGTGTTT
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Sense primer 1
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Note
GTGAGGCTCCCCTTTCTT
Sense primer 2 Anti-sense primer 2
GAGGTAAGCAAGCAGGACA GCAAGGAAAGGTGATAAAAGT
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Anti-sense primer 1
Letters shown in lowercase in SRP and LRP indicate the self-complementary stem of hairpin structure, while the fragments in bold are the half recognition site of Nt. BbvCI. The underlined portions denote the complementary fragments of target DNA. Notably, SRP was chemically tagged with a fluorophore of FAM and a quencher of DABCYL. The boxed segments in DPs, including DP1 and DP2, are complementary to the boxed one of LRP. NS1 and NS2 are the strands nicked and displaced from LRP and extended SRP respectively (see Scheme 1). MT1, MT2, MT3, and MT4 have the same base sequence as target DNA except for the point mutation in grey background, while
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NT was the target analogue randomly designed. When performing asymmetric PCR amplifications, sense primer 1/anti-sense primer 1 and sense primer 2/anti-sense primer 2 were used for p53
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amplicons and control amplicons, respectively.
2.2 Instruments
Fluorescence measurements were carried out at room temperature on a Hitachi F-7000
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fluorescence spectrometer (Hitachi, Ltd., Japan) with a Xenon lamp as the excitation source. The samples were excited at 492 nm, and the emission spectrum collection range was 500− 600 nm.
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Fluorescence intensity at 518 nm was used to evaluate the performance of the proposed strategy. The excitation and emission slits used in the experiments were both 5 nm and the photo- multiplier tube voltage was 600 V.
A 12% native polyacrylamide gel (native-PAGE) was freshly prepared and carried out on a gel
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electrophoresis instrument (BIO-RAD, USA) at a constant voltage of 80 V for 90 min by using 0.5× TBE buffer (4.5 mM Tris, 4.5 mM boric acid, 0.1 mM EDTA, pH 7.9) as the working buffer. The resulting gel was excited and imaged using the ChemiDoc XRS system with Image Lab analysis
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software (Bio- Rad). Considering SRP was FAM modified, sybr green I was only used in samples
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where SRP were not present, for example, DNA markers. 2.3 Two-wheel drive-based DNA nanomachine for target stimulus assay The assay experiment was carried out in 25-µL solution containing 15 µL of tris-buffer, 2 µL of SRP (5 µM), 2 µL of LRP (5 µM), 2µL of DP1 (5 µM), and 2 µL of target trigger (varying concentrations). Followed by incubation at 90
for 5 min and allowed to cool down to room
temperature. Then, 0.5 µL of Klenow fragment (3’→ 5’ exo-) polymerase, 0.5 µL of Nt. BbvCI nicking endonuclease, and 1 µL of 10 mM dNTPs were added into the above solution and incubated
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for 3.5 h at 37 . Finally, the reaction was inactivated by heating at 80 °C for 20 min, and the resulting mixture was used for the fluorescence measurement after adding 175 µL of dd-H2O.
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2.4 Genomic DNA extraction and asymmetric PCR amplifications Genomic DNA extraction from cultured human lung cancer cell line A549 was performed according to universal Genomic DNA exaction kit. Asymmetric PCR amplifications were carried out
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in terms of the process reported previously [30]. Briefly, 50 µL of 1× PCR buffer containing 1 µg of genomic DNA, 0.5 µM sense primer, 0.1 µM anti-sense primer, 0.15 mM dNTP mixture, as well as
denaturation step at 94
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2.5 U taq polymerase, was stirred thoroughly, and then moved to a PCR instrument. After a 5 min , the amplification was achieved by thermal cycling for 30 cycles at 94 °C
for 30 s, 55 °C for 30 s, 72 °C for 55 s, and a final extension at 72 °C for 7 min. The PCR products until required for gel electrophoresis and fluorescent assay.
3. Results and discussion
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were stored at 4
3.1 Design and working principle of the two-wheel drive-based DNA machine
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As shown in Scheme 1, two types of RP were designed and both engineered with two functional domains. One is a recognition region that is complementary to the target gene, which was at the 3’
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end of SRP or 5’ end of LRP. The other one is a half recognition site of restriction endonuclease Nt. BbvCI (shown in yellow), which was used to initiate the amplification machine. Besides, a DP was projected with several bases complementary to the 3’ end of LRP. Note that the SRP was tagged with a pair of fluorophore and quencher (FAM and DABCYL), in which the two molecules were in close proximity by the hairpin structure and the fluorescence of FAM was quenched in the initial state. The target DNA serving as a trigger acts as a template to align a pair of SRP and LRP perfectly along it,
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and the hairpin structure of LRP was deteriorated, thereby enabling the hybridization of LRP and DP (step
). With the aid of polymerase, the extension of 3’ end of SRP over target trigger and DP over
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LRP were implemented, during which the recognition site of Nt. BbvCI was formed in the latter one, causing nickase to specifically cleave the upper strand. As a result, a number of NS1 were generated by autonomously-performed nicking/polymerization/displacement cycles, and it seems like the ). Interestingly, the produced NS1 was
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operation was in a one-wheel drive manner (step
complementary to the extended part in SRP, so that it was able to compete with target DNA to
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hybridize with extended SRP. Although the binding force of NS1 to extended SRP was not powerful enough compared with target DNA, the driving force of polymerase would promote the hybridization event between NS1 and extended SRP, and lead to the replication of extended SRP (step
). The
distance between fluorophore and quencher was thus increased due to the hairpin structure opening,
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generating the fluorescence signal. Moreover, similarly, the recognition site of Nt. BbvCI was obtained in the newly formed duplex, and a large accumulation of NS2 was achieved (step
),
which was capable of hybridizing with SRP directly and made the fluorescence of FAM greatly ). This could lead to the second wheel driving. In addition, the target DNA released
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restore (step
was reused into next cycle (step
). We can see that the LRP-based wheel driving operation yields
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the primer to activate the extended SRP-based wheel driving like molecular nanomachine. In this sense, the two-wheel drive-based DNA nanomachine was successfully established, and two types of drives worked synergistically to sense target stimuli. Specially, since target DNA acts as the trigger species to induce the conformation change of LRP and sever as polymerization template for SRP (seen step
and
), the DNA nanomachine was not allowed to run completely when target
DNA was absent, indicating that no background was detected when used to screen target molecule.
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Scheme 1. Sketch of two-wheel drive based DNA nanomachine for target stimulus sensing. Binding to target stimulus;
LRP-based wheel drive and extension of SRP over target stimulus;
Binding of NS1 to extended SRP through competition with target stimulus; Hybridization of SRP with NS2.
Target stimulus recycling to initiate the
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based wheel drive;
Extended SRP
next round. The yellow segments inserted into oligonucleotide probes indicate the cleavage site of Nt.
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BbvCI.
3.2 Mechanism demonstration by gel electrophoresis
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To confirm that two-wheel drive based DNA nanomachine can operate as expected, gel electrophoresis was performed to give a straightforward characterization under optimized conditions (see Figure S1). As displayed in Figure 1, one single and obscure band corresponding to the designed SRP was observed in lane a, suggesting the fluorescence of FAM was quenched by DABCYL. Identical band was obtained in lane b where SRP, LRP, DP1 and polymerase coexisted, while two products with low mobility were observed in lane c upon addition of target trigger. Presumably, the upper band was the ternary complex of SRP-p53-LRP, and the lower band was the extended SRP/p53
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gene duplex. In contrast, two different bright bands with obvious shift in the positions in the presence of nickase were seen in lane e, and the band of SRP was totally disappeared. The mobility change
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and consumption of SRP represent that the nickase was worked, where the upper band in this lane was belong to the replicate helix of extended SRP, and the dominantly existed lower band was the hybridized complex of SRP and NS2 (see lane g). Notably, the relevant control sample in lane d was
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still the same as lane a, indicating that the DNA nanomachine was not operated in the absence of target trigger. So no any other background bands could be found. In addition, the NS1 mediated
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polymerization/nicking/displacement cycles on extended SRP was verified in lane f. In view of that the bands were identical to those in lane e, this phenomenon clearly exhibits that the extended SRP-based wheel-drive can be activated by the product of NS1 originating from the LRP-based
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wheel driving, strongly convincing the reasonability of proposed DNA nanomachine.
Figure 1. 12% native PAGE image of different mixtures. (a) SRP; (b) SRP+LRP+DP1+Polymerase; (c)
SRP+LRP+DP1+p53+Polymerase;
(d)
SRP+LRP+DP1+Polymerase+Nickase;
(e)
SRP+LRP+DP1+p53+Polymerase+Nickase; (f) SRP+p53+NS1+Polymerase+Nickase; and (g) SRP+NS2. “M” denotes the marker.
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3.3 Feasibility investigation for stimulus sensing In order to determine that the nanomachine based signal amplification can be utilized for stimulus
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sensing, different samples were firstly prepared and the corresponding fluorescent spectra were recorded as shown in Figure 2 to clarify this point. One can see that the signal of the DNA nanomachine (curve b) in the absence of target trigger was much closed to the inherent fluorescence
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of SRP (curve a), suggesting the negligible background fluorescence. Excitingly, a significantly increased fluorescence (about 800 %) was detected when encountering with the target trigger (curve
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c), indicating it possess powerful capability for target analyte analysis. As a control test, the mixture in the absence of nickase (curve d) or polymerase (curve e) compared with line c was also performed. As anticipated, these samples could not give any detectable fluorescence increase and closely approaches to the background. The data yielded by this study provides confirmatory evidence that
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the DNA nanomachine is available for target sensing.
Figure 2. Typical fluorescent emission spectra collected from the solutions containing SRP (a), SRP+LRP+DP1+Polymerase+Nickase
(b),
SRP+LRP+DP1+p53+Polymerase+Nickase
(c),
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SRP+LRP+DP1+p53+Polymerase (d), SRP+LRP+DP1+p53+Nickase (e). The signal increase was deduced from [(F-F0)/F0], where F and F0 are the data in the presence and absence of target trigger, respectively. The concentrations of SRP, LRP, and DP1 were 500 nM, while the involved target was
3.4 Interrogating capability for target stimulus
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100 nM.
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Once possessing the ability to response to certain trigger, the sensing capability of two-wheel drive-based DNA nanomachine was verified. Figure 3A depicts the spectral data collected for various
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target concentrations ranging from 0 to 15 nM. We can see that the maximal value was obtained at 8 nM, and further increasing the target concentration would not give higher signal. Since the target DNA at 1 pM can induce a detectable signal (see inset), we define it as the detection limit. Figure 3B is the calibration curve deduced from the peak values acquired at 518 nm, and the regression
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equation is Y= 387.36+ 292.41X (R2= 0.9949) associated with a dynamic range from 1 pM to 8 nM, where Y and X represent the peak value and target trigger concentration, respectively. Undoubtedly, the excellent performance was attributed to uniquely designed signal amplification and greatly
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reduced background. In contrast to reported approaches that relying on fluorescence as output signal
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[31-36], the assay ability was apparently improved regarding to the detection limit.
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Figure 3. (A) Fluorescent spectra upon addition of various concentrations of target stimulus. The curves from bottom to top correspond to the target DNA with concentrations of 0, 1 pM, 10 pM, 100 pM, 400 pM, 700 pM, 1 nM, 2 nM, 3 nM, 6 nM, 8 nM, 10 nM and 15 nM, respectively. Inset
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amplified the curves at low target concentrations range. (B) Corresponding calibration plot obtained from the peak values at 518 nm over the range of 1 pM to 8 nM. Error bars are the standard deviations of three parallel experiments.
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Essentially, the proposed DNA nanomachine works on the basis of isothermal nucleic acid amplification. This amplification technique is a simple process that rapidly and efficiently
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accumulates nucleic acid sequences at constant temperature. Since the early 1990s, a large number of isothermal amplification systems have been described as alternatives to polymerase chain reaction (PCR). A recent pioneered review literature [37] has summarized the development of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration,
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encompassing the previous studies presented in the past two decades. An inspiring fact is that, in terms of the limit of detection, linear response range and the ability to discriminate single base mismatch, the present DNA nanomachine is superior to considerable number of fluorescent assay
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systems based on the isothermal nucleic acid amplification (see the details in Table S1).
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Despite the impressive progress made in the development of fluorescent sensing systems, however, when compared with some other transducing techniques (e.g., colorimetric assay [11, 38] and electrochemical measurements [39, 40]), the assay sensitivity is not powerful enough and still needs to be further improved to meet the demand for ultra-sensitive detection of nucleic acids. According to our own research experiences [38] and literature reports [41-43], several strategies could be employed to improve further the assay sensitivity, for example, the optimization of the base sequence of SRP and LRP [41-42] and adjustment the concentrations of oligonucleotide probes [43]. We think
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that increasing the proportion of “C-G” pairs in the stem of SRP would make FAM and DABCYL closer to each other, leading to the lower background fluorescence. In addition, since the background
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fluorescence was smart circumvented except for the inherent fluorescence of SRP, increasing the ratio of SRP/LRP would enhance further the output signal when profiling certain concentration of target stimulus. The optimizing experiments are expected to be capable of improving further the
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two-wheel drive-based DNA nanomachine sensitivity. 3.5 Stimuli specificity study
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To our knowledge, a majority of polymorphisms in the human genome are caused by mutations that involve one or more nucleotides, and the sequence-specificity detection is of great importance [44]. To evaluate whether single nucleotide mutation could be correctly distinguished from the right one with the present DNA nanomachine, four synthetic target molecules, each with a single
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nucleotide mutation in different positions were used for specificity study at the same concentration. Notably, the mutation points in MT1 and MT2 were designed at the junction site, while MT3 and MT4 were arbitrarily projected. As revealed in Figure 4, the target stimulus (wild-type p53 gene)
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could be easily differentiated from other analogues no matter where the mutation point was located, permitting the facile distinction of known sequence variants differing by only one single nucleotide.
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Importantly, the responses induced by the single nucleotide mutation at the junction sites were relatively weak, indicating the impressive progress in the detection of single nucleotide mutation. Although the mutant target molecules were likely to capture one pair of SHP and LHP, they are unable to drive efficiently the machine operate owing to the decreased matching ability. Especially when the mutant nucleotides were at the junction sites, the mismatched single nucleotide is incapable of leading to the extension of SRP over MT1 or prohibits the polymerization of NS1 over extended
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SRP that is obtained from MT2 as polymerization template. Additionally, we found that the randomly planned non-target trigger only induces a negligible fluorescence increase.
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Actually, DNA nanomachines are required to be specifically initiated by the stimulus [23]. However, it’s still a biggest challenge to detect specifically nucleic acids that differ only in a single nucleotide [45], and genetic diagnosis was currently limited by the cumbersome nature of some DNA
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sensing methods. For instance, the capacity of conventional molecular beacon is relative poor in the detection of single nucleotide substitution [46-48], while the ligation-based strategies utilizing RCA
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for mutation analysis often involved time-consuming operations, such as denaturing PAGE purification, despite of the desired specificity by high-fidelity DNA ligase [41, 49]. In contrast, the proposed DNA nanomachine was easily constructed and the corresponding specificity at the junction site was comparable to ligation-based strategies [49-51] but higher than many literature values [23,
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46, 48, 52, 53].
Figure 4. Assay specificity of the DNA nanomachine toward 100 nM target stimuli. MT1, MT2, MT3, and MT4 are single-nucleotide mutations, and NT is randomly designed oligonucleotide strand with same length as target stimulus. The relative signal response was calculated as [(F-F0)/ (Ft-F0)],
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where F0, Ft, and F are the values corresponding to samples of blank, p53 target, and MTs (MT1, MT2, MT3, and MT4) or NT, respectively. The data are presented as means ± standard deviation.
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3.6 Real samples detection Essentially, all the advancements of technology should be directed toward the practical applications, and strategies holding certain perspective in real biological samples are highly
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appreciated. Therefore, the aim of this section was to study whether the proposed DNA nanomachine has the practical potential for analyzing target molecule in genomic DNA. In light of that the DNA
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extracted from cancer cells was double-stranded, asymmetric PCR amplifications were performed first to produce corresponding single-stranded DNAs, and the results of gel electrophoreses are shown in Figure 5A. One can see that a detectable band corresponding to p53 amplicons and control amplicons was observed in lane a and lane b, respectively, indicating the successful amplifications.
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The produced single-stranded target stimulus and control stimulus could not be seen since the poor embedding ability of sybr Green I toward single-stranded DNA. Figure 5B are the spectra recorded from the asymmetric PCR amplification products. Compared with the control amplicons in which the
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value (curve b) is closed to the blank (curve a), a remarkable signal response was obtained by target
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amplicons (curve c), offering robust capability to monitor the presence of particular sequence from genomic DNA. Inset displays the specific fluorescent difference after deducting the background value, confirming further that the DNA nanomachine is adoptable for genomic DNA detection. These measured data imply practical potential for clinical relevant applications.
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Figure 5. (A) Gel electrophoresis analysis of asymmetric PCR amplification products. Lane a, p53
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amplicons; lane b, control amplicons. (B) Fluorescent spectra of two-wheel drive based DNA nanomachine for blank sample (a), control amplicons (b) and p53 target amplicons (c). Inset: comparative data on the signal triggered by different amplicons. F and F0 are the peak values in the presence and absence of amplicons, respectively.
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4. Conclusion
In aggregate, a two-wheel drive-based DNA nanomachine was herein proposed as a proof-of-concept to develop an impressive DNA nanodevice for the detection of cancer-related gene.
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This DNA molecular machine possesses three highlighted characteristics: (1) The two-wheel
and
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drive-based DNA nanomachine induce two consecutive stages of fluorescence amplification (step ), and target stimulus was recycled (step
), achieving a substantially amplified readout
signal; (2) This nanomachine uses target DNA as fuel to cause LRP-based wheel drive to communicate with extended SRP based drive, while the usually brought background in the absence of stimulus was entirely circumvented; (3) The protocol is highly specific due to the stringently designed amplification pathway, which allows easy discrimination of sequences with one nucleotide mutation. In such a situation, the two-wheel-drive-based DNA nanomachine was a progressive
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achievement in material science, molecular computing, and biosensing, offering applicable routes
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toward achieving versatile and powerful molecular machines.
Acknowledgments
This work was supported by Ministry of Science and Technology of China (2015CB931804),
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National Natural Science Foundation of China (NSFC) (grant# 81273548, 21275002, and U1505225), and Independent Research Project of State Key Laboratory of Photocatalysis on Energy
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and Environment (NO. 2014CO1).
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