Nucleic Acid Amplification-Free Bioluminescent Detection of

Subscriber access provided by Binghamton University | Libraries

Article

Nucleic Acid Amplification-Free Bioluminescent Detection of MicroRNAs with High Sensitivity and Accuracy Based on Controlled Target Degradation Qinfeng Xu, Fei Ma, Si-qiang Huang, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Nucleic Acid Amplification-Free Bioluminescent Detection of MicroRNAs with High Sensitivity and Accuracy Based on Controlled Target Degradation Qinfeng Xu,b,† Fei Ma,a,† Si-qiang Huang,c Bo Tanga,* and Chun-yang Zhanga,* a

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, 250014, China. b

College of Food and Biological Engineering, Shaanxi University of Science and Technology,

Xi’an, 710021, China c

Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen,

518055, China * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected].

Tel.:

+86

0531-86180010;

Fax: +86

[email protected].

1

ACS Paragon Plus Environment

0531-86180017;


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Accurate and sensitive detection of microRNAs is crucial to clinical diagnosis and therapy. Most of microRNA assays require target conversion in combination with nucleic acid amplification to improve the detection sensitivity, which may compromise the assay accuracy and specificity. Herein we report a sensitive bioluminescent method for microRNA assay on the basis of controlled target degradation without target conversion and nucleic acid amplification. In this assay, the target microRNA can be specifically degraded by exonuclease III after hybridization to its complementary probe, releasing adenosine monophosphate (AMP) from microRNA itself. The AMP then triggers an efficient bioluminescence generation system to produce a strong bioluminescence signal. This assay is highly sensitive with zero-background signal and a detection limit of 7.6 fM even without target amplification, and it can discriminate the single-nucleotide difference among microRNA family members with extremely high discrimination ratio. With the assistance of magnetic separation to eliminate the interference of endogenous ATP, ADP and AMP in sample matrix, this assay can be further applied to absolute quantification of microRNAs in cancer cells and tissues from lung cancer patients, holding great potential in biomedical research and clinical diagnosis.

2


Page 2 of 26

Page 3 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MicroRNAs are short non-coding RNA molecules which play important roles in the control of cell development and physiology1,2 through regulating the expression of complementary messenger RNAs.3,4 MicroRNAs are closely associated with various pathological processes,5,6 and the deregulation of endogenous microRNA expression has been found in many human diseases including heart disease,7 hepatocellular carcinoma,8 breast cancer,9 ovarian cancer,10 and lung cancer.11 Consequently, microRNAs are now regarded as emerging biomarkers for disease diagnosis,12 prognosis13 and treatment,14 and there is an urgent demand to develop efficient microRNA assays.15 Northern blotting is the standard method for microRNA assay,16 but it suffers from large sample consumption, being time-consuming and semi-quantitative. In addition, the sensitivity of Northern blotting (in the nanomole range) is unsatisfactory for the detection of low abundant microRNA targets.17 To improve the sensitivity, a series of nucleic acid amplification approaches have been introduced. Among them, the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is widely used,18 in which the microRNA target is converted to complementary DNA (cDNA) and then amplified for quantification by PCR.19 To eliminate the use of expensive thermal cyclers in PCR-based assay, various isothermal nucleic acid amplification techniques have been developed as the alternatives to PCR,20 including strand displacement amplification (SDA),21 exponential isothermal amplification (EXPAR),22,23 rolling circle amplification (RCA)24 and loop-mediated isothermal amplification (LAMP).25 The general strategy used in these amplification-based assays is the conversion of target microRNAs to DNA sequences followed by nucleic acid amplification. Even though these amplification approaches provide powerful platforms for 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

highly sensitive detection of microRNA, the inaccurate target conversion and nonspecific amplification may lead to false-positive results and high background signal, which may compromise the assay accuracy and specificity. In some cases, complicated experimental procedures and data processing are required to eliminate the nonspecific background signal. Consequently, nucleic acid amplification-free methods with improved sensitivity are highly required for accurate and sensitive microRNA quantification. In this research, we develop a sensitive bioluminescent method for microRNA assay on the basis of controlled target degradation without the involvement of any nucleic acid amplification (i.e., nucleic acid amplification-free). This assay is very sensitive with a detection limit of 7.6 fM even without target amplification, and it can discriminate the single-nucleotide difference among microRNA family members with high discrimination ratio. Moreover, this assay can be used to absolutely quantify microRNAs in cancer cells and tissues from lung cancer patients with the assistance of magnetic separation technique.

EXPERIMENTAL SECTION Materials. The oligonucleotides used in this research were listed in Table 1. The DNA probes, DEPC-treated water and NaCl were obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). The synthetic RNA targets, dCTP and recombinant RNase inhibitor were bought from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Dynabeads® M-270 streptavidin (MB), UltraPure™ DNase/RNase-free distilled water, 1 M Tris-HCl buffer (pH 7.5) and the ATP determination kit were purchased from Invitrogen (USA). Exonuclease III (Exo III), Exonuclease T (Exo T), T7 exonuclease 4


Page 4 of 26

Page 5 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(T7 Exo), RNase I, RNase H, ATP sulfurylase, Cutsmart buffer and bovine serum albumin (BSA) were obtained from New England BioLabs (Beverly, MA, USA). Phospho(enol)pyruvic

acid

monosodium

salt

hydrate

(PEP),

adenosine

5´-phosphosulfate (APS), pyruvate kinase from rabbit muscle (PK) and adenylate kinase (AK) from chicken muscle were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The 96-well white microplate was bought from Fisher Scientific (Pittsburgh, PA, USA). The paraffin-embedding lung tissue samples of non-small cell lung cancer (NSCLC) patients and healthy persons were obtained from the affiliated hospital of Guangdong Medical University (Zhanjiang, China), and the experiments were approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University. The following buffer solutions were prepared with DEPC-treated water. Solution A (0.1 M NaOH and 0.05 M NaCl) and 2× binding & washing buffer (20 mM Tris-HCl, 2 mM EDTA and 200 mM NaCl, pH 7.5) were used for coupling the biotinylated DNA probes to the streptavidin-modified MB. Table 1. Sequences of the synthesized oligonucleotidesa note

sequence (5’-3’)

let-7a

UGA GGU AGU AGG UUG UAU AGU U

let-7b

UGA GGU AGU AGG UUG UGU GGU U

let-7c

UGA GGU AGU AGG UUG UAU GGU U

miR-21

UAG CUU AUC AGA CUG AUG UUG A

miR-205

UCC UUC AUU CCA CCG GAG UCU G

let-7a probe

AAC TAT ACA ACC TAC TAC CT*C* A* 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

let-7a probe-biotin

AAC TAT ACA ACC TAC TAC CTC A-biotin

miR-21 probe-biotin

TCA ACA TCA GTC TGA TAA GCT A-biotin

miR-205 probe-biotin CAG ACT CCG GTG GAA TGA AGG A-biotin a

The asterisk indicates the phosphorothioate modification.

Bioluminescent Detection of MicroRNAs. For microRNA hydrolysis, target let-7a was mixed with different nucleases in 1× reaction buffer (NEB buffer 3 or cutsmart buffer) in a total volume of 10 µL, and then the reaction proceeded at room temperature or 37 °C for half an hour. For microRNA-DNA duplex hydrolysis, all conditions were identical except that microRNA-DNA duplex formed in 2× reaction buffer for half an hour prior to being mixed with the enzymes. After the hydrolysis, 2.5 µL of reaction mixture was added into the ATP detection system for bioluminescent assay in a total volume of 50 µL. The ATP detection system was consisted of 1× Cutsmart buffer, 4.0 µL of the AMP-to-ATP conversion buffer (1.0 µL of 1 U/µL AK, 1.0 µL of 1 U/µL PK, 1.0 µL of 10 mM dCTP, and 1.0 µL of 4.8 mM PEP), 6.0 µL of pyrophosphate-to-ATP conversion buffer (5.0 µL of 1.0 µM APS, 2.5 µL of 1.0 mU ATP sulfurylase) and 5 µL of ATP detection buffer (0.5 mM D-luciferin, 1.5 µg/mL firefly luciferase, 5 mM MgSO4, 1 mM DTT, 100 µM EDTA, and 25 mM Tricine buffer (pH 7.8)). After the above reagents were mixed, the bioluminescence signal was monitored by a Glomax luminometer (Promega, Madison, WI, USA) at room temperature.

6


Page 6 of 26

Page 7 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Quantification of MicroRNAs with the Assistance of Magnetic Separation. The magnetic beads were washed with the same volume of solution A (0.1 M NaOH and 0.05 M NaCl) and 2× binding & washing buffer (20 mM Tris-HCl, 2 mM EDTA and 200 mM NaCl, pH 7.5) for twice, respectively. Then an equal volume of 5’-biotin-modified probes (10 µM) was incubated with the magnetic beads in 1× binding & washing buffer at room temperature for 20 min with gentle mixing. After the binding procedure, the conjugates were separated with a magnet and the supernatant was removed. Finally, the conjugates were washed three times with 1× binding & washing buffer, resuspended and stored at 4 °C for further use. The DNA probe-modified MB and the appropriate amount of synthetic microRNA target or RNA extracted from the cells were mixed in an Eppendorf tube containing 1× binding & washing buffer, 0.1 mg/mL BSA and RNase inhibitor (40 U) in a total volume of 100 µL. The hybridization mixture was incubated at room temperature for 1 hour with gentle rotation. Then the MB was separated with a magnet and washed twice with 500 µL of 1× binding & washing buffer (containing 0.1% Tween 20). Finally, the washed beads were incubated in 40 µL of 1× cutsmart buffer containing 0.3 U/µL Exonuclease III and 0.1 U/µL Exo T at room temperature for 1 hour. After the hydrolysis, all reaction mixtures were added into the ATP detection system for bioluminescent assay in a total volume of 50 µL. Quantification of MicroRNAs Extracted from Cells and Tissues. Cell lines were cultured with 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50 U/mL penicillin and 50 mg/mL streptomycin in a 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

humidified chamber containing 5% CO2 at 37 °C. Cells were collected with trypsinization, washed twice with ice-cold PBS (pH 7.4), and pelleted at 300× g at 25 °C for 5 min. About 3 × 106 ~ 4 × 106 cells was collected for total RNA extraction using miRNeasy Mini Kit (Qiagen, German) according to the manufacturer’s handbook. Alternatively, the miRNeasy FFPE kit (Qiagen, Germany) was used when total RNA was extracted from the formalin-fixedly paraffin-embedded tissue samples. The concentrations of total RNA were determined by measuring the absorbance at 260 nm, 230 nm and 280 nm with a spectrophotometer. Quantitative polymerase chain reaction (qPCR) was employed for the quantification of miRNA using NCode™ EXPRESS SYBR® Green ER™ miRNAqRT-PCR Kit (Invitrogen, USA) in a BIO-RAD CFX connectTM Real-Time system.

RESULTS AND DISCUSSION Principle of Bioluminescent Detection of MicroRNAs. The principle of the proposed assay is illustrated in Scheme 1. The 3’-phosphorothioate-modified (Scheme 1, blue color) DNA probe (Scheme 1, green color) is fully complementary to the target microRNA (Scheme 1, red color). The presence of target microRNA leads to the formation of microRNA-DNA duplex. Afterwards, the microRNA strand in the duplex may be specifically degraded from its 3´-hydroxyl termini by Exo III to release 5’-GMP, 5’-CMP, 5’-TMP and 5’-AMP, while the DNA probes are resistant to the digestion of Exo III due to the phosphorothioate modification.26 The released AMP may be converted to ATP in the presence of PEP and dCTP by coupling the enzymatic reactions of 8


Page 8 of 26

Page 9 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AK and PK, and subsequently the resultant ATP may be converted to AMP, producing a strong bioluminescence signal with the assistance of firefly luciferase and luciferin. The released AMP may subsequently induce the cyclic AMP pyrophosphorylation-ATP depyrophosphorylation for the generation of an enhanced bioluminescence signal (Scheme 1).27-29 Notably, in this assay, the output signal is originated from the target microRNA itself without the involvement of any target conversion or nucleic acid amplification, thus efficiently eliminating the false-positive result and exhibiting significant advantage over EXPAR which is very prone to template-independent amplification.30 Unlike RT-PCR with the involvement of thermocycling, this assay may be carried out under isothermal condition. Moreover, the efficient bioluminescence generation system guarantees the accuracy and sensitivity of the assay. Scheme 1. Illustration of bioluminescent detection of microRNAs based on controlled target degradation.

Optimization of Experimental Conditions. Exo III exhibits strong preference for double-stranded DNA,31 and it can also efficiently degrade the RNA strand in DNA-RNA hybrids.

31-35

Thus, Exo III is used in this research for specific degradation

of microRNA. Fig. 1a-b shows the bioluminescent monitoring the hydrolysis of 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

single-stranded microRNAs (Figure 1a) and microRNA-DNA hybrids (Figure 1b) in response to different RNA hydrolysis nucleases, including RNase I, Exo T, T7 Exo and Exo III. No significant bioluminescence signal is observed in response to RNase I and RNase H because of their incapability to release 5’-AMP from microRNA.36,37 In contrast, the addition of Exo T to single-stranded microRNAs induces a distinct bioluminescence signal (Figure 1a). Interestingly, only the addition of T7 Exo and Exo III to the microRNA-DNA hybrids may induce distinct bioluminescence signals (Figure 1b). These results are consistent with the previous reports that Exo T, T7 Exo and Exo III can degrade RNA to release 5’-AMP.31,38,39 Notably, T7 Exo and Exo III generate relative weak bioluminescence signals compared with Exo T due to the simultaneous degradation of both RNA and DNA from microRNA-DNA hybrids in the 3’ →5’ direction for Exo III and in the 5’ → 3’ direction for T7 Exo, which may lead to the incomplete hydrolysis of microRNA-DNA hybrids by T7 Exo and Exo III. To solve this issue, we designed a phosphorothioate-modified DNA probe to protect the DNA terminal from being hydrolyzed by T7 Exo and Exo III, ensuring that only microRNA can be hydrolyzed by T7 Exo and Exo III (Figure 1c). Fig. 1d indicates that Exo III may achieve much higher hydrolysis efficiency than T7 Exo under the identical experimental conditions. Therefore, Exo III and 3’-phosphorothioate-modified DNA probes were used in subsequent experiments to accomplish complete sequence-specific degradation of target microRNAs.

10


Page 10 of 26

Page 11 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Bioluminescent monitoring the degradation of microRNAs by different nucleases. (a) bioluminescent monitoring the hydrolysis of single-stranded microRNAs in response to different nucleases; (b) bioluminescent monitoring the hydrolysis of microRNA-DNA hybrids in response to different nucleases; (c) phosphorothioate modification of DNA probe (marked by red) at 3’-end (3’-S) and 5’-end (5’-S), respectively;

(d)

bioluminescent

monitoring

the

hydrolysis

of

microRNA-phosphorothioate-modified DNA hybrids by Exo III and T7 Exo, respectively.

3’-S

(red)

and

5’-S

(blue)

indicate

the

DNA

probes

with

phosphorothioate modification at 3’-end and 5’-end, respectively. The controls without phosphorothioate modification are shown in black. Error bars represent the standard deviation of 3 independent experiments.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Feasibility of MicroRNA Assay. To demonstrate the feasibility of the proposed assay, let-7a microRNA was used as the model target. The let-7a microRNA is deregulated in various tumor types.40,41 In the presence of let-7a target (0.25 pM), the probe (2.0 pM) and Exo III (0.3 U/µL), the let-7a may be specifically degraded by Exo III, resulting in a strong bioluminescence signal (Figure 2). On the contrary, in the absence of any one of above three components, no degradation of let-7a occurs and no significant bioluminescence signal is observed with zero-background signal. The obtained zero-background signal may be ascribed to the efficient elimination of both incorrect target conversion and nonspecific nucleic acid amplification in this assay.19,22,42

Figure 2. Bioluminescent detection of microRNA in the presence of probe + Exo III (No let-7a), let-7a + Exo III (No probe), let-7a + probe (No Exo III), and let-7a + probe + Exo III (All added), respectively. The let-7a concentration is 0.25 pM, and the probe concentration is 2.0 pM, and the Exo III concentration is 0.3 U/µL. Error bars represent the standard deviation of 3 independent experiments.

12


Page 12 of 26

Page 13 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Detection Sensitivity. Our goal is to develop a highly sensitive assay even without the involvement of any nucleic acid amplification. To demonstrate the sensitivity of the proposed assay, we measured the bioluminescence signal induced by let-7a at various concentration. A linear correlation is obtained between the bioluminescence intensity and the let-7a concentration in the range from 25 fM to 1 pM (Figure 3). The regression equation is B = 183.5 ＋ 36.33 × C with an extremely high correlation coefficient of 0.9994, where C represents the let-7a concentration and B represents the bioluminescence intensity. The limit of detection (LOD) is estimated to be 7.6 fM. Notably, the sensitivity of current assay has enhanced by 4 orders of magnitude compared with that of RCA-based method,43 and more than 1 order of magnitude compared with that of EXPAR-based method,44 even without the involvement of any target conversion and nucleic acid amplification. The high sensitivity of this assay is attributed to the extremely low background signal and the high efficient bioluminescence generation system.

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

Figure 3. Linear correlation between the bioluminescence intensity and the let-7a concentration. Error bars represent the standard deviation of 3 independent experiments.

Detection Specificity. The specificity of the assay is of great importance to accurate quantification of target microRNA, and is greatly challenged by the high similarity of microRNA sequence. To evaluate the selectivity of the proposed method, we quantitatively measured the let-7 microRNA family members with high sequence homology including let-7a, let-7b and let-7c (Figure 4a). Because let-7a is perfectly matched to the probe, its signal is significantly higher than those of let-7b and let-7c, and the discrimination ratio is 2.28 between let-7a and let-7b, and 1.44 between let-7a and let-7c (Figure 3b, top part). Notably, the improved selectivity can be achieved by using the optimal hydrolysis temperature due to different melting temperature of microRNA-probe duplex. When the temperature is elevated to 59 °C, the discrimination ratio is 71.42 between let-7a and let-7b, and 13.15 between let-7a and let-7c

(Figure

4b,

bottom

part),

much

higher

than

that

of

nucleic

acid

amplification-based assay (usually below 3).45 The improved discrimination ratio may be ascribed to the disruption of the mismatched let-7b-probe duplex and let-7c-probe duplex at 59 °C. In contrary, the perfectly matched let-7a-probe duplex remains intact at 59 °C and may be subsequently degraded by Exo III for the generation of a strong bioluminescence signal. These results indicate that this assay can successfully discriminate microRNAs with high sequence homology. 14


Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (a) Sequences of let-7a, let-7b and let-7c. (b) Bioluminescent detection of let-7a, let-7b and let-7c under reaction temperature of 37 °C (top) and 59 °C (bottom). Error bars represent the standard deviation of 3 independent experiments.

Magnetic Separation-Assisted Assay. It should be noted that the endogenous ATP, ADP and AMP in real sample matrix may generate a high background signal.46 We introduced magnetic separation technique to solve this issue. The 3’ biotin-modified DNA probes are assembled to the streptavidin-modified magnetic beads (MB) through specific biotin-streptavidin interaction (Scheme 2). The target microRNAs can be magnetically separated from the endogenous ATP, ADP and AMP via specific binding to the DNA probes. After separation, the microRNAs in microRNA-DNA probe-MB complex may be degraded by Exo III and monitored by luminescent assay as shown in Scheme 1. 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

Scheme 2. Schematic illustration of the magnetic separation-based microRNA assay.

Multiplex

Detection

of

MicroRNAs.

We

further

applied

this

magnetic

separation-assisted assay for multiplexed detection of three different microRNAs including let7-a, miR-21 and miR-205. When let-7a probe is used, only let-7a generates a distinct bioluminescence signal, while both miR-21 and miR-205 produce negligible signals (Figure 5). Similarly, only miR-21 generates a distinct bioluminescence signal in the presence of miR-21 probe, and only miR-205 generates a distinct bioluminescence signal in the presence of miR-205 probe (Figure 5). Moreover, a linear correlation is obtained between the bioluminescence intensity and the target concentration in the range from 25 fM to 1 pM for three microRNAs, and the regression equation is B = 311.66 ＋ 31.08 × C for miR-21, B = 287.60 ＋ 28.04 × C for let-7a, and B = 379.17 ＋ 15.05 × C for miR-205, respectively, where C represents the microRNA concentration and B represents the bioluminescence intensity. These results suggest that the magnetic separation-assisted assay can be applied for 16


Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


accurate quantification of different microRNAs. Notably, the more the adenines contained in the microRNA sequences (the number of adenines in miR-21, let-7a and miR-205 sequences are 6, 5 and 3, respectively, for current assay), the higher the bioluminescence signal being obtained (Figure 5). Consequently, the detection sensitivity is directly proportional to the number of adenines in the microRNA sequence, which is confirmed by the improved sensitivity induced by the addition of a poly (A) tail (~250 adenosines) for microRNA assay.47

Figure 5. (a) Mutiplexed detection of let7-a, miR-21 and miR-205 using the coressponding MB-DNA probes. (b) A linear correlation is obatined between the bioluminescence intensity and the microRNA concentration. Error bars represent the standard deviation of 3 independent experiments. 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Real Sample Analysis. To investigate whether this method can be applied for real sample analysis, we quantified miR-21 in H460 cancerous cells. After extraction of total RNA samples by miRNeasy Mini Kit (Qiagen, German), the concentration of miR-21 is determined to be 7.27 fmol/10 µg total RNA (Figure 6a). Notably, this result is consistent with the standard qRT-PCR analysis, in which the miR-21 concentration is quantified to be 7.21 fmol/10 µg total RNA. We further measured the target microRNA in clinical samples from lung cancer patients. Previous study has shown that miR-205 is over-expressed in non-small cell lung cancer (NSCLC).48 Our results demonstrate that the expressions of miR-205 obtained from tumor tissues of lung cancer patients are significantly higher than those obtained from the normal tissues (Figure 6b, blue bars, paired t test, P = 0.014), in good accordance with the standard qRT-PCR assay (Figure 6b, red bars), suggesting that the proposed method has the capability to absolutely quantify microRNAs extracted from human cancer cells and tissues with excellent accuracy.

18


Page 18 of 26

Page 19 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (a) Quantification of miR-21 extracted from H460 cancerous cells. (b) Quantification of lung cancer-related miR-205 in tumor and normal tissues using the proposed bioluminescent method (blue bars) and qRT-PCR (red bars), respectively. Error bars represent the standard deviation of 3 independent experiments.

CONCLUSION In summary, we have developed a simple and sensitive bioluminescent method for microRNA assay based on controlled target degradation. The output signal is originated from the microRNA itself without the involvement of any target conversion and nucleic acid amplification, and high accuracy can be achieved with zero 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

background signal. This assay exhibits high sensitivity with a detection limit of as low as 7.6 fM, and it can discriminate the single-nucleotide difference among microRNA family members with extrembly high discrimination ratio. Importantly, this assay can be applied to absolute quantification of microRNAs in human cancer cells and tissues from lung cancer patients with the assistance of magnetic separation. This assay may provide an excellent platform for simple, rapid and cost-effective quantification of microRNAs, holding a great potential for further applications in biomedical research and clinical diagnosis.

AUTHOR INFORMATION Corresponding Authors *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. ORCID Chun-yang Zhang: http://orcid.org/0000-0002-8010-1981 Bo Tang: http://orcid.org/0000-0002-8712-7025 Author Contributions †

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS 20


Page 20 of 26

Page 21 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This research was financially supported by the National Natural Science Foundation of China (Grant Nos. 1325523, 21527811 and 21405169), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Ambros, V. Nature 2004, 431, 350-355. (2) Bartel, D. P. Cell 2009, 136, 215-233. (3) Lim, L. P.; Lau, N. C.; Garrett-Engele, P.; Grimson, A.; Schelter, J. M.; Castle, J.; Bartel, D. P.; Linsley, P. S.; Johnson, J. M. Nature 2005, 433, 769-773. (4) He, L.; Hannon, G. J. Nat. Rev. Genet. 2004, 5, 522-531. (5) Rachagani, S.; Kumar, S.; Batra, S. K. Cancer Lett. 2010, 292, 8-16. (6) O'Connell, R. M.; Rao, D. S.; Chaudhuri, A. A.; Baltimore, D. Nat. Rev. Immunol. 2010,

10, 111-122. (7) Ikeda, S.; Kong, S. W.; Lu, J.; Bisping, E.; Zhang, H.; Allen, P. D.; Golub, T. R.; Pieske, B.; Pu, W. T. Physiol. Genomics 2007, 31, 367-373. (8) Yamamoto, Y.; Kosaka, N.; Tanaka, M.; Koizumi, F.; Kanai, Y.; Mizutani, T.; Murakami, Y.; Kuroda, M.; Miyajima, A.; Kato, T. Biomarkers 2009, 14, 529-538. (9) Iorio, M. V.; Ferracin, M.; Liu, C.-G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Ménard, S.; Palazzo, J. P.; Rosenberg, A.; Musiani, P.; Volinia, S.; Nenci, I.; Calin, G. A.; Querzoli, P.; Negrini, M.; Croce, C. M. Cancer Res. 2005, 65, 7065-7070. (10) Taylor, D. D.; Gercel-Taylor, C. Gynecol. Oncol. 2008, 110, 13-21. (11) Takamizawa, J.; Konishi, H.; Yanagisawa, K.; Tomida, S.; Osada, H.; Endoh, H.; Harano, T.; Yatabe, Y.; Nagino, M.; Nimura, Y.; Mitsudomi, T.; Takahashi, T. Cancer Res. 2004, 64, 3753-3756. (12) Kosaka, N.; Iguchi, H.; Ochiya, T. Cancer Sci. 2010, 101, 2087-2092. 22


Page 22 of 26

Page 23 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Slack, F. J.; Weidhaas, J. B. New Engl. J. Med. 2008, 359, 2720-2722. (14) Garzon, R.; Calin, G. A.; Croce, C. M. Annu. Rev. Med. 2009, 60, 167-179. (15) Dong, H.; Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. Chem. Rev. 2013, 113, 6207-6233. (16) Goda, S. K.; Minton, N. P. Nucleic Acids Res. 1995, 23, 3357. (17) Lee, H.; Srinivas, R. L.; Gupta, A.; Doyle, P. S. Angew. Chem. Int. Ed. 2015, 54, 2477-2481. (18) Kroh, E. M.; Parkin, R. K.; Mitchell, P. S.; Tewari, M. Methods 2010, 50, 298-301. (19) Resnick, K. E.; Alder, H.; Hagan, J. P.; Richardson, D. L.; Croce, C. M.; Cohn, D. E.

Gynecol. Oncol. 2009, 112, 55-59. (20) Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Chem. Rev. 2015, 115, 12491-12545. (21) Tian, T.; Xiao, H.; Zhang, X.; Peng, S.; Zhang, X.; Guo, S.; Wang, S.; Liu, S.; Zhou, X.; Meyers, C.; Zhou, X. Chem. Commun. 2013, 49, 75-77. (22) Jia, H.; Li, Z.; Liu, C.; Cheng, Y. Angew. Chem. Int. Ed. 2010, 49, 5498-5501. (23) Ma, F.; Yang, Y.; Zhang, C. Y. Anal. Chem. 2014, 86, 6006-6011. (24) Jonstrup, S. P.; Koch, J.; Kjems, J. RNA 2006, 12, 1747-1752. (25) Li, C.; Li, Z.; Jia, H.; Yan, J. Chem. Commun. 2011, 47, 2595-2597. (26) Xu, Q. F.; Cao, A. P.; Zhang, L. F.; Zhang, C. Y. Anal. Chem. 2012, 84, 10845-10851. (27) Xu, Q.; Zhu, G.; Zhang, C. Y. Anal. Chem. 2013, 85, 6915-6921. (28) Xu, Q. F.; Zhang, Y.; Zhang, C. Y. Chem. Commun. 2015, 51, 5652-5655. (29) Lee, H. J.; Ho, M. R.; Tseng, C. S.; Hsu, C. Y.; Huang, M. S.; Peng, H. L.; Chang, H. Y.

Anal. Biochem. 2011, 418, 19-23. (30) Tan, E.; Erwin, B.; Dames, S.; Ferguson, T.; Buechel, M.; Irvine, B.; Voelkerding, K.; 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Niemz, A. Biochemistry 2008, 47, 9987-9999. (31) Rogers, S. G.; Weiss, B. In Methods Enzymol.; Academic Press, 1980; pp 201-211. (32) Richardson, C. C.; Lehman, I. R.; Kornberg, A. J. Biol. Chem. 1964, 239, 251-258. (33) Keller, W.; Crouch, R. Proc. Natl. Acad. Sci. U. S. A. 1972, 69, 3360-3364. (34) Roychoudhury, R.; Wu, R. J. Biol. Chem. 1977, 252, 4786-4789. (35) Weiss, B.; Rogers, S. G.; Taylor, A. F. In DNA Repair Mechanisms, Friedberg, E. C.; Fox, C. F., Eds.; Academic Press, 1978; pp 191-194. (36) Spahr, P. F.; Hollingworth, B. R. J. Biol. Chem. 1961, 236, 823-831. (37) Donis-Keller, H. Nucleic Acids Res. 1979, 7, 179-192. (38) Deutscher, M. P.; Marlor, C. W.; Zaniewski, R. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 4290-4293. (39) Shinozaki, K.; Tuneko, O. Nucleic Acids Res. 1978, 5, 4245-4261. (40) Jeong, H. C.; Kim, E. K.; Lee, J. H.; Yoo, H. N.; Kim, J. K. Mol. Med. Report. 2011, 4, 383-387. (41) Takamizawa, J.; Konishi, H.; Yanagisawa, K.; Tomida, S.; Osada, H.; Endoh, H.; Harano, T.; Yatabe, Y.; Nagino, M.; Nimura, Y. Cancer Res. 2004, 64, 3753-3756. (42) Li, Y.; Liang, L.; Zhang, C.-y. Anal. Chem. 2013, 85, 11174-11179. (43) Harcourt, E. M.; Kool, E. T. Nucleic Acids Res. 2012, e65. (44) Wang, G. L.; Zhang, C. Y. Anal. Chem. 2012, 84, 7037-7042. (45) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.; Xia, F. J.

Am. Chem. Soc. 2013, 135, 4604-4607. (46) Kimmich, G. A.; Randles, J.; Brand, J. S. Anal. Biochem. 1975, 69, 187-206. 24


Page 24 of 26

Page 25 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(47) Fang, S. P.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044-14046. (48) Markou, A.; Tsaroucha, E. G.; Kaklamanis, L.; Fotinou, M.; Georgoulias, V.; Lianidou, E. S. Clin. Chem. 2008, 54, 1696-1704.

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only

26


Page 26 of 26

Nucleic Acid Amplification-Free Bioluminescent Detection of

Recommend Documents