A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR

Aug 27, 2018 - A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRT-PCR) for Precise Quantitation of Messenger RNA Mutation in ...
0 downloads 0 Views 2MB Size
Download PDF
Subscriber access provided by Kaohsiung Medical University

Article

A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRTPCR) for Precise Quantitation of Messenger RNA Mutation in Single Cells Yuanyuan Sun, Hui Tian, Chenghui Liu, Dandan Yang, and Zhengping Li ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00524 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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 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 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.

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 14 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

ACS Sensors

A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRT-PCR) for Precise Quantitation of Messenger RNA Mutation in Single Cells Yuanyuan Sun, Hui Tian, Chenghui Liu,* Dandan Yang, Zhengping Li* Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China. *Corresponding author. Tel/Fax: +86 29 81530859. E-mail: [email protected]; [email protected] ABSTRACT: Precise detection of the low copy numbers of messenger RNA (mRNA) mutation in single cells is of great significance but still remains challenging. Herein, by integrating the outstanding features of a rationally designed peptide nucleic acid (PNA) clamp for highly selective discrimination of single-nucleotide variation, and droplet digital PCR for ultrasensitive and precise quantification, we have developed a robust one-step droplet digital reverse transcription PCR (ddRT-PCR) method which enables precise mRNA mutation detection in single cells with ultrahigh specificity to clearly discern as low as 0.01% mutated mRNA in a high background of wild-type mRNA. In virtue of its outstanding single-molecule level sensitivity and ultrahigh specificity, this ddRT-PCR method holds great promise for studying cellular heterogeneity at the single cell level, as well as for the precise quantification of mutant mRNAs in complex plasma or serum for liquid biopsy.

Keywords: mRNA mutation, PNA clamp, ddPCR, single cell analysis, analytical methods Messenger RNA (mRNA) plays a critical role in gene expression by serving as the bridge to translate the genetic codes of DNA into functional proteins.1 More and more evidence has revealed that site-specific mRNA mutations, which directly determine the function of translated proteins, are closely associated with the development of diverse diseases including cancers. Therefore, tumorrelated mRNA mutations have become greatly appreciated in recent years as specific biomarkers to assess tumor cell migration in the organism or in the bloodstream.2-6 For example, EGFR mutation testing in tissue or blood samples has been clinically approved for non-small cell lung cancer patients, which at the same time works as predictive marker for the selection of EGFR tyrosine kinase inhibitors.7-11 It should be noted that the clinically meaningful mutant mRNAs are typically in quite low abundances in heterogeneous clinical samples, which necessitates highly sensitive and specific methods to precisely quantify the trace amount of mutated mRNA sequences in a huge pool of wild sequences. More importantly, mRNA mutation-driven tumorigenesis often begins in rare cells within a tissue or organism. Since the mutant mRNA levels may vary substantially between the cancer and normal cells, and even in cell populations bearing the same genotype, the precise and sensitive detection of mutated mRNA transcripts in individual single cells may bring deep insight to elucidating the complexity and heterogeneity of cancer development.12-16 However, single cell mRNA mutation analysis still remains a big challenge because it requires ultrahigh sensitivity, preferentially at the single-molecule level.

Up to now, the real-time quantitative reverse transcription-PCR (qRT-PCR) is the most widely used standard methods for mRNA analysis.17,18 One main limitation of qRT-PCR is its low selectivity in heterogeneous biosamples where the high background of wild sequences may mask the detection of low copy numbers of mutant ones. For example, the well-optimized allele-specific PCRs can only detect about 1% mutation frequency.18 Moreover, the sensitivity and accuracy of the traditional qRT-PCR are difficult to precisely quantify the copy number of mRNA target at the single-cell level.19 Therefore, the development of a mRNA mutation assay which enables precise target quantitation in single cells with ultrahigh selectivity to clearly discriminate singlebase mutated target in a vast number of wild sequences, is greatly desired. Herein, with the assistance of a rationally-designed peptide nucleic acid (PNA) clamp, we wish to report a robust one-step droplet digital RT-PCR (ddRT-PCR) assay which enables precise mRNA mutation detection at the single-cell and single-molecule level with ultrahigh selectivity to discriminate as low as 0.01% mutated mRNA in a high background of wild-type sequences.

EXPERIMENTAL SECTION Reagents and materials. The DNA primers, RNase-free water, Taq Hot Start DNA polymerase, Ribonuclease Inhibitor, dNTPs (10 mM), and the plasmids for preparing the target BRAF mRNA were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). SuperScript™ IV Reverse Transcriptase (SSIV) and TaqMan probe were obtained from Thermo Fisher Scientific (WA, USA). The PNA was synthesized by Panagene (Daejeon, Korea). The

ACS Paragon Plus Environment

ACS Sensors 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

ddPCR Supermix for Probes was obtained from Bio-Rad (USA). The detailed sequences of nucleic acids involved in this work were all listed in Table S1. The QX100 Droplet Digital PCR Platform (ddPCR) (Bio-Rad, USA) was used for precise quantitative detection of target mRNA. RNase-free water was used to prepare all of the reaction solutions in this work. In order to make the detection results more convincing, a pair of 299-nt target mRNA fragments of BRAF gene, including a wild-type sequence (wtRNA) and a singlenucleotide mutated sequence (mutRNA, BRAF V600E mutation), were employed as the proof-of-concept targets in this work (see detailed sequences in Table S1). At first, the synthesized mutant and wild target gene of BRAF were constructed into the pBluescript II SK(+) cloning vector with the recognition site of restriction endonuclease Hind III by Takara Biotechnology. Then both mutRNA and wtRNA were obtained by T7 transcriptase-assisted in vitro transcription. The experimental protocols were typically as follows. Firstly, the plasmid of the mutRNA was digested by the Hind III at 37 °C for 16 hours and the enzymedigested product was separated and purified by agarose electrophoresis analysis. Afterwards, in vitro transcription reaction was performed with a Takara in vitro Transcription T7 Kit at 42 °C for 2 hours following the manufacturer's protocols. The transcriptional product was processed by multiple DNase treatments at 37 °C for 60 min and purified with a Takara MiniBEST Universal RNA Extraction Kit. Finally, the product of mutRNA was quantified on a Nanodrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA). The wtRNA was also obtained following the same procedures. The agarose electrophoresis analysis with 2% (w/v) agarose gels was carried out to characterize the products of in vitro transcription. The results shown in Figure S1 clearly demonstrated that the 299-nt mutRNA and wtRNA were both successfully prepared, respectively. Standard procedures for the detection of mutRNA with the ddRT-PCR assay. The ddRT-PCR reaction was performed in a single-step in a total 20 μL mixture containing 500 nM primers (containing forward and reverse primer), 500 nM PNA, 250 nM Taqman probe, 20 U Ribonuclease inhibitor, 0.5 mM dNTPs, 80 U SSIV reverse ranscriptase, reverse transcription (RT) buffer, supermix and target mRNAs (or cell lysate). Firstly, the reverse primer, RT buffer, 0.5 mM dNTPs and target mRNAs (or cell lysate) were mixed, heated for 3 min at 80 °C, and then incubated for 5 min at 60 °C. Afterward, the mixture was put on ice, and the forward primer, PNA, Taqman probe, Ribonuclease inhibitor, reverse transcriptase (SSIV), supermix were further introduced to make a final 20 μL volume for ddRT-PCR. Then eight such prepared mixtures (each 20 μL, containing varying concentrations of mRNA target) and 70 μL volume of droplet generation oil (BioRad) were transferred, respectively, into the sample well and oil well of DG8TM droplet generator cartridge (BioRad). The loaded cartridge was covered with a gasket and placed in the droplet generator, where the samples were partitioned by the oil within the microchannels of the cartridge to create up to 20 000 stable nanoliter-sized water-in-oil droplets.

Page 2 of 14

All the nucleic acid sequences, supermix, reverse transcriptase (SSIV) and dNTPs were water-soluble, so they could be encapsulated and partitioned in the internal aqueous phase of the monodispersed water-in-oil droplets. Afterwards, the droplets were placed in a 96-well PCR plate, and then the plate was sealed with foil by employing a PX1 PCR Plate Sealer (Bio-Rad) and moved into a T100 Thermal Cycler for subsequent RT-PCR in every droplets. The ddRT-PCR was performed with the following procedures: 30 min at 58 °C, then heated for 5 min at 94 °C, followed by 45 cycles of 94 °C for 20 s, 67 °C for 20 s and 60 °C for 1 min, 98 °C for 10 min. Following the completion of such RT-PCR reaction, the droplets were seriatim detected by QX100 Droplet Reader (Bio-Rad). The acquired data were analyzed by the Quantasoft analysis software (Bio-Rad) with the automatic threshold setting. dMIQE checklist (Table S2) supplies more details for the proposed ddRT-PCR assay. Cell culture and the mutRNA detection in extracted total RNA or in single cell lysate. The human melanoma cell line (SK-MEL-28) was purchased from the American Type Culture Collection (ATCC), and cervical cancer cells (Hela) was obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China), which were all maintained in DMEM Medium replenished with 100 μg/mL streptomycin, 100 U/mL penicillin and 10% (v/v) fetal calf serum. The thyroid cancer cell (ARO) was purchased from Beina Chuanglian Biotechnology Institute (Beijing, China) and cultured in RPMI 1640 Medium containing 10% (v/v) fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. All of them were cultured at 37 °C under an appropriate humid atmosphere containing 5% CO2. The total RNA samples were extracted from the correspondingly cultured cells with Trizol reagent (Invitrogen, Beijing, China) following the manufacturer’s description, and the amount of extracted total RNAs were quantified with a Nanodrop 2000 UV-Vis Spectrophotometer, respectively. The mutRNA levels in the total RNA samples extracted either from SK-MEL-28 cells or Hela cells were both determined by the ddRT-PCR method according to the standard procedures stated above. As for the mutRNA quantification in individual single cells, a single SK-MEL-28 cell, or a single Hela cell, or single ARO cells were directly obtained from a massive dilution of cells suspension. After the digestion with trypsin, the cells were firstly centrifuged for 5 min with 700 rpm to remove the cell culture medium, washed and resuspended with PBS. 50 μL of sufficiently diluted cell suspension was placed on a glass slide, and individual single cells can be easily captured with a Narishige micromanipulator system equipped on an inverted microscope. Each of the selected single cells was heated at 95 °C for 3 min, and the lysate was directly used as the sample to proceed the ddRT-PCR mutRNA assay following the standard experimental procedures mentioned above.

RESULTS AND DISCUSSION Principle of the PNA clamp-based ddRT-PCR assay for the detection of mRNA mutation. The principle of the PNA clamp-based ddRT-PCR strategy is schematically illustrated in Figure 1. In this work, the mutant BRAF

ACS Paragon Plus Environment

Page 3 of 14 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

ACS Sensors

mRNA (a 299-nt long fragment, see detailed sequence in Table S1), where a base U in the wild-type sequence (wtRNA) is mutated to A in the mutant mRNA (mutRNA, BRAF V600E mutation), is selected as a proof-of-concept target. This site-specific mutation is closely associated with the melanoma, the deadliest form of skin cancer.20,21 As illustrated in Figure 1, a 16-nt PNA sequence, which exhibits excellent ability to distinguish singe-base variation,5,22-25 is designed to be complementary to the wtRNA with a melting temperature (Tm) of ~70 ºC. In contrast, the PNA/mutRNA duplex with a A/A mismatch site in the middle of the sequence has an approximately 10~20 ºC lower Tm than that of the PNA/wtRNA.

Figure 1. Illustration of the PNA clamp-based ddRT-PCR assay for the detection of mRNA mutation. (a) All of the reaction components required for both reverse transcription and PCR amplification are put into a single tube to be partitioned into a large number of droplets, most of which contains either 1 or 0 copy mRNA molecule according to poisson distribution. The PNA is capable of tightly clamping the wtRNA to completely inhibit subsequent RT-PCR reaction so that the droplet containing wtRNA will be fluorescence-negative. In contrast, PNA cannot clamp the mutRNA so that the RT-PCR reaction towards mutRNA can be normally proceeded, which produces

fluorescence-positive droplets. (b) After RT-PCR reaction, the droplet mixture is detected by Droplet Reader and the data are analyzed for precise quantification of mutRNA.

Firstly, all the reaction components consisting of the PNA, PCR primers, TaqMan probe, mRNA targets, reverse transcriptase (SSIV) and the supermix (containing polymerase and dNTPs) are all mixed together and then partitioned into water-in-oil droplets. It is worth noting that the mRNA targets are divided randomly into the droplets following Poisson distribution. If the copy number of mRNA target is extremely low, each droplet will contain either 0 or 1 mRNA molecule. Then, the reverse transcription (RT) and PCR reaction are both proceeded separately in individual droplets.26-28 Under the optimized DNA extension conditions (58~60 ºC) in this study, in the droplets containing wtRNA, the PNA can pair with the perfectly complementary wtRNA with high stability. As such, the wtRNA will be tightly clamped by the PNA so that the reverse transcription as well as the subsequent PCR can be completely inhibited. Nevertheless, in the droplets containing the mutRNA target, the PNA cannot clamp the mutRNA and the RT-PCR amplification towards mutRNA can be proceeded normally. Therefore, in this PNA clampbased ddRT-PCR assay, the RT-PCR reaction accompanied with the turn-on fluorescence of TaqMan probe can only occur in the droplets containing mutRNA, which can be finally identified as the fluorescence-positive droplets. Meanwhile, the droplets with wtRNA or without any RNA sequences will be fluorescence-negative. The positive and negative droplets are finally counted by QX100 droplet reader with QuantaSoft software to analyze the data for precise quantification of mutRNA (Figure 1b). The critical role of PNA clamp for the discrimination of single-base variation. As depicted in Figure 1, the ability of PNA for the highly selective discrimination of single-base difference is most critical for the proposed mRNA mutation assay. The polyacrylamide gel electrophoresis (PAGE) results shown in Figure 2 clearly testify that the PNA can only efficiently clamp the wtRNA, while the RT-PCR reaction towards mutRNA will not be influenced. One can see that for the mutRNA, no matter whether PNA is present (lane 7) or not (lane 5), the bands corresponding to the PCR products (128 bp) can be clearly identified with almost the same pixel intensities, indicating that the PNA cannot clamp mutRNA to inhibit subsequent RT-PCR. For the wtRNA, the distinct band of the PCR products can be also observed if PNA is absent (lane 9). Nevertheless, upon the introduction of PNA, the band of PCR products disappears completely (lane 11), suggesting that wtRNA is tightly clamped by PNA so that it can no longer template subsequent RT-PCR. All of these results have clearly verified the excellent performance of PNA clamp to discriminate the single-nucleotide difference between mutRNA and wtRNA, which can guarantee the high specificity of the proposed mRNA mutation assay.

ACS Paragon Plus Environment

ACS Sensors 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 2. Non-denaturing polyacrylamide gel electrophoresis (PAGE) results of the RT-PCR products in the presence or absence of PNA clamp, by using mutRNA or wtRNA as the template, respectively. Lane 1: double-strand DNA markers; lane 2: forward primer only; lane 3: reverse primer only. Lane 4~lane 11 shows the products of different components after RT-PCR reaction and the corresponding components are clearly shown below the PAGE image. The concentrations of mutRNA and wtRNA are both 10 fM.

Analytical performance of the clamp-based ddRTPCR strategy for the detection of mutRNA. Some important parameters including the temperatures for reverse transcription and PCR annealing-extension, PNA concentration and the PCR thermal cycle number, which may affect the sensitivity and specificity of the proposed ddRT-PCR assay for mutRNA detection, are all investigated and optimized (see Figure S2-S5). Finally, 58 ºC for reverse transcription, 60 ºC for PCR annealing-extension, 500 nM PNA clamp and 45 thermal cycles are selected as the optimal conditions. Under such selected conditions, the analytical performance of the proposed ddRT-PCR assay is primarily assessed by detecting series dilutions of the mutRNA. As displayed in Figure 3a and Figure S6a, with the increase of the mutRNA dosage from 1 aM to 100 fM, the corresponding positive droplet numbers are gradually increased. The positive droplets produced by as low as 1 aM mutRNA target (equal to 12 copies) can be distinctly discerned from that of blank control, suggesting an ultrahigh sensitivity of the proposed method at the singlemolecule level. Furthermore, when the logarithm (log) of positive droplets numbers (N) are plotted against the log of mutRNA concentrations (CmutRNA/M), as can be seen from Figure 3b, a good linear relationship is obtained in the range of 1 aM to 10 fM, spanning over 4 orders of magnitude. The linear regression equation is log N = 14.97 + 0.7837 log CmutRNA/M with a correlation coefficient R2 of 0.9947. For comparison, the wtRNA is also detected with the same reaction conditions. As demonstrated in Figure 3c and Figure S6b, no positive droplets can be observed even with 1 fM wtRNA, further proving the high selectivity of the proposed method for mutRNA analysis.

Page 4 of 14

Figure 3. Analytical performance of the ddRT-PCR assay for the detection of mRNA. (a) The positive droplet numbers produced by mutRNA with varying concentrations. The concentrations of mutRNA from left to right are 0, 1 aM, 10 aM, 100 aM, 1 fM, 10 fM and 100 fM, respectively; (b) The linear relationship between the log of the positive droplet numbers and the log of the concentrations of mutRNA. Error bars represent the standard deviation of three independent measurements; (c) The positive droplet numbers produced by different concentrations of wtRNA under the same experimental conditions for mutRNA detection. The concentrations of wtRNA from left to right are 0, 1 aM, 10 aM, 100 aM and 1 fM, respectively.

Furthermore, the analytical performance of the proposed ddRT-PCR assay, such as the sensitivity, accuracy and precision, is compared with the widely used real-time quantitative RT-PCR (qRT-PCR) method by using the same primer/PNA set. The results displayed in Figure S7 clearly indicate that the detection limit of mutRNA by using ddRTPCR is 10-fold lower than that of qRT-PCR method. More importantly, the ddRT-PCR method possesses much higher detection precision and accuracy than qRT-PCR for precise determination of mutRNA target with slight concentration variations (Figure S8). Therefore, it can be concluded that the ddRT-PCR strategy shows much better reliability than qRT-PCR for the accurate and precise detection of mutRNA, particularly when the target is of extremely low copy numbers. Evaluation of the specificity of the clamp-based ddRT-PCR strategy for mutRNA analysis. Besides for the ultrahigh sensitivity, a practical mRNA mutation assay should also possess high specificity which enables accurate determination of extremely low copy numbers of mutRNA in a high background of wtRNA, particularly for the detection of mutRNA in complex clinical biosamples. In this work, to investigate the specificity of the ddRT-PCR method, mutRNA is mixed with wtRNA at different ratios varying from 0 to 100% with a fixed total RNA concentration of 10 fM. Such mixtures directly serve as the samples of the ddRT-PCR assay to evaluate its specificity. It can be seen from Figure 4a and Figure S9 that the number of positive droplets rises gradually with the increasing ratio of mutRNA in the RNA mixture. The log of positive droplet numbers (N) is linearly proportional to the log of proportion of mutRNA in the range of 0.01% ~100%. It is

ACS Paragon Plus Environment

Page 5 of 14 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

ACS Sensors

worth noting that the pure 10 fM wtRNA (0% mutRNA) can only produce negligible less than 5 positive droplets, so the gradually increased positive droplets are undoubtedly aroused by mutRNA target in the RNA mixtures. Fascinatingly, one can see from Figure 4 and Figure S9 that the proposed ddRT-PCR assay can clearly discern as low as 0.01% mutRNA target, which is equivalent to a quite low concentration of 1 aM or 12 molecules, in a pool of large number of wtRNA, indicating an ultrahigh selectivity towards the detection of one-base mutated mRNA.

Figure 4. Evaluation of the specificity of the ddRT-PCR assay for mutRNA detection. (a) Positive droplet numbers produced by the mixture of mutRNA/wtRNA in different proportions (the total concentration of the RNA mixture is fixed at 10 fM). From left to right, the proportion of mutRNA in the mixture is 0%, 0.01%, 0.1%, 1%, 10%, 50% and 100%, respectively; (b) The linear relationship between the log of the positive droplet numbers and the log of the proportions of mutRNA in the RNA mixtures. Error bars represents the standard deviation of three repetitive measurements.

Conventional methods for the detection of mRNA mutation are widely based on the ability of PCR to discriminate the mutation site of the reverse-transcribed cDNAs. However, the well-optimized allele-specific PCRs can only approximately detect mutations at frequencies of about 1%.18 Recently, the ligation chain reaction (LCR)based in vitro assay and the ligation-assisted rolling circle amplification (RCA)-based fluorescence imaging method are respectively developed by our group and other group for the detection of mRNAs.29,30 Although the ligases are inherently sensitive to base variations, a single-base mutation can lead to ~18.6% and ~1% interference for the detection of normal mRNA, respectively. Therefore, the specificity of the ddRT-PCR method for the detection of mRNA with a single-base variation is superior to most of the existing mRNA assays. The superior specificity should be ascribed to the excellent ability of PNA for highly selective clamping of wtRNA. According to the principle illustrated in Figure 1, even if the reverse transcription process of wtRNA is not completely inhibited by the PNA clamp, the very small amount of wild-type cDNAs will be also efficiently clamped by PNA during subsequent PCR process, guaranteeing the ultrahigh specificity of the proposed method for quantification of mutRNA. Detection of mutRNA in complex cell extracts and serum. Sharing the distinct advantages of outstanding single-molecule level sensitivity and ultrahigh specificity to detect as low as 0.01% mutation frequency, the proposed ddRT-PCR method may offer a powerful and reliable tool for the precise quantification of mutated mRNA in complex biosamples. In order to test the capability of the proposed

method for mRNA analysis in real biological samples, the amount of mutRNA in the total RNAs extracted from two kinds of cancer cells, melanoma cancer cells (SK-MEL-28) and cervical cancer cells (Hela), are determined respectively. According to previous literatures,31 the target BRAF mRNA in SK-MEL-28 cells are all mutated, while only wild-type BRAF mRNAs are present in Hela cells, which is also verified by the sequencing results (Figure S10). As shown in Figure S11, the mutRNA target in as little as 10 pg SK-MEL-28 total RNA can be obviously detected, while for the Hela total RNA, even the presence of 20 ng RNA material cannot produce any positive droplets, further verifying the ultrahigh sensitivity and specificity of the ddRT-PCR assay. The amount of mutRNA content in 2 ng SK-MEL-28 total RNA is determined to be 180 aM (calculated in the 20 μL reaction mixture). When 2 ng of the same batch total RNA sample is pre-spiked with 600 aM of standard mutRNA target, the average amount of mutRNA in such spiked sample is determined to be 828 aM with a recovery of 108% by five parallel experiments. These results prove that the ddRT-PCR method is applicable for mutRNA analysis in complex real samples. To further validate its potential clinical applicability, the proposed ddRT-PCR mRNA assay was also applied to the detection of spiked mutRNA in the serum from a healthy volunteer in our laboratory. The results shown in Figure S12 clearly demonstrate that the complex serum matrixes will not interfere with the mutRNA analysis and thus the proposed method is of great potential for detecting low levels of mutRNA in clinical blood/serum samples. Precise determination of mutRNA in individual single cells. As mentioned above, increasing knowledge about the association between cellular mRNA expression heterogeneity and complex diseases highlights the demand for accurate and precise mRNA detection at the single cell level. Finally, we have examined the performance of the ddRT-PCR assay for mutRNA detection in individual single cells. First of all, individual single cells are respectively picked up by using a Narishige micromanipulator system and lysed by heating at 95 ºC for 3 min. The mutRNA level in the lysate of each cell is independently determined by the ddRT-PCR assay. The detection results of mutRNA in 13 randomly captured single SK-MEL-28 cells are displayed in Figure 5a and Figure S13a. One can see that no positive droplet is produced by the blank control, while the positive droplets aroused by the 13 single SK-MEL-28 cells can be all distinctly observed. The determined copy number of mutRNA in each cell varies from 18 to 46. Moreover, the average copy number of mutRNA in the detected individual cells (∼28 copies per cell) is consistent with the average values determined in the lysates of 10 cells (∼23 copies per cell) or 100 cells (∼27 copies per cell), respectively. By contrast, as shown in Figure 5b and Figure S13b, no any positive droplet is generated for individual Hela cells because no mutant BRAF mRNA is present in such cells. Furthermore, the proposed PNAassisted ddRT-PCR strategy is further applied to the detection of mutRNA in individual heterozygous (BRAF V600E mutation) ARO thyroid cancer cells in which both mutRNA and wtRNA are expressed.32 In this study, in order to avoid the influence of cell-to-cell variation of mutRNA level in

ACS Paragon Plus Environment

ACS Sensors 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 cells, two ARO cells were captured and lysed, and half of the lysate was tested by the PNA clamp-based ddRT-PCR in which only the mutRNA can produce positive droplets. Meanwhile, the other half of the lysate was interrogated by ddRT-PCR without adding PNA clamp so that the total amount of mutRNA and wtRNA were determined together. The results displayed in Figure 5c clearly demonstrate that the PNA clamp-assisted ddRT-PCR strategy is feasible for detecting low levels of mutRNA in a background of wtRNA in individual single cells. All of the above results clearly prove that the ddRT-PCR strategy is extremely promising for the reliable, accurate and precise mutRNA detection in individual single cells due to its ultrahigh sensitivity and specificity.

Page 6 of 14

Meanwhile, excellent specificity is also achieved which allows the clearly discrimination of as low as 0.01% mutRNA target in a high background of wtRNA. It is worth noting that circulating cell-free nucleic acids have been regarded as potential cancer markers for the liquid biopsy, which can avoid the invasive tumor tissue biopsies.33 Notably, recent studies have demonstrated that circulating mRNAs, which can also stably exist in the bloodstream, may serve as promising cancer markers.5,33-36 Since the copy numbers of cellular mRNAs are much higher than those of the corresponding DNAs, it is believable that in some cases, the detection of cancer-related circulating mRNA mutation in plasma or serum may be more promising and reliable for cancer diagnosis and management than the detection of cell-free DNAs.5,33-36 Although the applicability of the ddRT-PCR assay in clinical bioliquid samples is not tested in this study because the clinical samples from melanoma skin cancer patients with definite diagnosis are quite difficult to collect for us, we believe that due to its single-molecule level sensitivity and superior specificity, this robust PNA clamp-based ddRTPCR assay may serve as a powerful tool for the accurate and precise determination of extremely low copy numbers of mutRNA in single cells or in complex blood samples, which holds great promise for the study of cellular heterogeneity at the single cell level, and for the liquid biopsy-related biomedical research by using mRNA mutation as the biomarker.

ASSOCIATED CONTENT

Figure 5. Determination of mutRNA in individual single cells. (a) The positive droplet numbers produced by 13 independent single SK-MEL-28 cells; (b) The positive droplet numbers produced by 8 individual Hela cells; (c) Detection of mutRNA in heterozygous single ARO cells. It should be noted that in the presence of PNA clamp, only the mutRNA can arouse positive droplets (red bars), while in the absence of PNA, the positive droplets (black bars) are produced by both the mutRNA and wtRNA.

Supporting Information. The sequences of oligonucleotides, detailed information for the ddRT-PCR mutRNA assay according to the guidelines of the dMIQE checklist, detailed optimization of experimental conditions in the ddRT-PCR, the comparison between the ddRT-PCR assay and qRT-PCR method, sequencing results of the BRAF mRNA in total RNA samples extracted from human cancer cells, and the determination of mutRNA level in total RNA extracted from cancer cells or in the serum are presented in Electronic Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

In addition, the results shown in Figure S15 clearly indicate that if a few numbers of SK-MEL-28 cells, even only a single cell, coexist in a large pool of Hela cells, the SK-MEL-28 cell-responsive positive droplets can all be clearly observed, and the presence of abundant Hela cells does not interfere with the detection of SK-MEL-28 cells. Therefore, the proposed ddRT-PCR method also shows great promise for the detection of rare cells with mutated mRNA in a large number of normal cells.

AUTHOR INFORMATION

CONCLUSIONS

Notes

Corresponding Author

Email:* [email protected]; [email protected] Phone/Fax: +86 29 81530859 Author Contributions

All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

In summary, with an elegantly designed PNA clamp, a versatile ddRT-PCR approach is proposed for the ultrasensitive and specific detection of low copy numbers of mutRNA. Both the reverse transcription reaction and PCR amplification are accomplished in a single mixture without any sample transfer operation, which is less likely to cause contamination. This ddRT-PCR assay exhibits an ultrahigh sensitivity, which allows the accurate detection of as low as 1 aM mutRNA target at the single cell level.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21335005, 21622507, 21472120), Program for Changjiang Scholars and Innovative Research Team in University (IRT 15_R43), and the Fundamental Research Funds for the Central Universities (GK201802016).

REFERENCES

ACS Paragon Plus Environment

Page 7 of 14 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

ACS Sensors

(1) Li, G. W.; Xie, X. S. Central Dogma at the Single-Molecule Level in Living Cells. Nature 2011, 475, 308-315. (2) Taniguchi, K.; Kajiyama, T.; Kambara, H. Quantitative Analysis of Gene Expression in a Single Cell by qPCR. Nat. Methods 2009, 6, 503-506. (3) Huber, F.; Lang, H. P.; Backmann, N.; Rimoldi, D.; Gerber, C. Direct Detection of a BRAF Mutation in Total RNA from Melanoma Cells Using Cantilever Arrays. Nat. Nanotech. 2013, 8, 125-129. (4) Huang, J.; Wang, H.; Yang, X.; Quan, K.; Yang, Y.; Ying, L.; Xie, N.; Ou, M.; Wang, K. Fluorescence Resonance Energy TransferBased Hybridization Chain Reaction for in situ Visualization of Tumor-Related mRNA. Chem. Sci. 2016, 7, 3829-3835. (5) Das, J.; Ivanov, I.; Montermini, L.; Rak, J.; Sargent, E. H.; Kelley, S. O. An Electrochemical Clamp Assay for Direct, Rapid Analysis of Circulating Nucleic Acids in Serum. Nat. Chem. 2015, 7, 569-575. (6) Tang, Y.; Zhang, X.; Tang, L.; Yu, R.; Jiang, J. In situ Imaging of Individual mRNA Mutation in Single Cells Using LigationMediated Branched Hybridization Chain Reaction (LigationbHCR). Anal. Chem. 2017, 89, 3445-3451. (7) van Zandwijk, N.; Mathy, A.; Boerrigter, L.; Ruijter, H.; Tielen, I.; de Jong, D.; Baas, P.; Burgers, S.; Nederlof, P. EGFR and KRAS Mutations as Criteria for Treatment with Tyrosine Kinase Inhibitors: Retro- and Prospective Observations in Non-small-cell Lung Cancer. Ann. Oncol. 2007, 18, 99-103. (8) Mok, T.; Wu, Y. L.; Lee, J. S.; Yu, C. J.; Sriuranpong, V.; Sandoval-Tan, J.; Ladrera, G.; Thongprasert, S.; Srimuninnimit, V.; Liao, M.; Zhu, Y.; Zhou, C.; Fuerte, F.; Margono, B.; Wen, W.; Tsai, J.; Truman, M.; Klughammer, B.; Shames, D. S.; Wu, L. Detection and Dynamic Changes of EGFR Mutations from Circulating Tumor DNA as a Predictor of Survival Outcomes in NSCLC Patients Treated with First-line Intercalated Erlotinib and Chemotherapy. Clin. Cancer Res. 2015, 21 , 3196-3203. (9) Yuan, J. Q.; Zhang, Y. L.; Li, H. T.; Mao, C. EGFR Mutation Testing in Blood for Guiding EGFR Tyrosine Kinase Inhibitor Treatment in Patients with Nonsmall Cell Lung Cancer. Medicine 2017, 96, e6151. (10) Sheikine, Y.; Rangachari, D.; McDonald, D. C.; Huberman, M. S.; Folch, E. S.; VanderLaan, P. A.; Costa, D. B. EGFR Testing in Advanced Non-Small-Cell Lung Cancer, A Mini-Review. Clin. Lung Cancer 2016, 17, 483-492. (11) Thomas J. Lynch, Daphne W. Bell, , Raffaella Sordella, Sarada Gurubhagavatula, Ross A. Okimoto, Brian W. Brannigan, Patricia L. Harris, Sara M. Haserlat, Jeffrey G. Supko, Frank G. Haluska, David N. Louis, David C. Christiani, Jeff Settleman, and Daniel A. Haber. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to Gefitinib. New Engl. J. Med. 2004, 350, 2129-2139. (12) Meacham, C. E.; Morrison, S. J. Tumour Heterogeneity and Cancer Cell Plasticity. Nature 2013, 501, 328-337. (13) Larsson, C.; Grundberg, I.; Soderberg, O.; Nilsson, M. In situ Detection and Genotyping of Individual mRNA Molecules. Nat. Methods 2010, 7, 395-397. (14) Pollen, A. A.; Nowakowski, T. J.; Shuga, J.; Wang, X.; Leyrat, A. A.; Lui, J. H.; Li, N.; Szpankowski, L.; Fowler, B.; Chen, P.; Ramalingam, N.; Sun, G.; Thu, M.; Norris, M.; Lebofsky, R.; Toppani, D.; Kemp, D. W., 2nd; Wong, M.; Clerkson, B.; Jones, B. N.; Wu, S.; Knutsson, L.; Alvarado, B.; Wang, J.; Weaver, L. S.; May, A. P.; Jones, R. C.; Unger, M. A.; Kriegstein, A. R.; West, J. A. Low-Coverage Single-Cell mRNA Sequencing Reveals Cellular Heterogeneity and Activated Signaling Pathways in Developing Cerebral Cortex. Nat. Biotechnol. 2014, 32, 1053-1058. (15) Zong, C.; Lu, S.; Chapman, A. R.; Xie, X. S. Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell. Science 2012, 338, 1622-1626. (16) Rakszewska, A.; Stolper, R. J.; Kolasa, A. B.; Piruska, A.; Huck, W. T. Quantitative Single-Cell mRNA Analysis in Hydrogel Beads. Angew. Chem. Int. Ed. 2016, 55, 6698-6701.

(17) Bustin, S. A. Quantification of mRNA Using Real-Time Reverse Transcription PCR (RT-PCR): Trends and Problems. J. Mol. Endocrinol. 2002, 29, 23-29. (18) Chen, W. W.; Balaj, L.; Liau, L. M.; Samuels, M. L.; Kotsopoulos, S. K.; Maguire, C. A.; Loguidice, L.; Soto, H.; Garrett, M.; Zhu, L. D.; Sivaraman, S.; Chen, C.; Wong, E. T.; Carter, B. S.; Hochberg, F. H.; Breakefield, X. O.; Skog, J. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles. Mol. Ther.Nucl. Acids 2013, 2, e109. (19) Shi, J.; Zhou, M.; Gong, A.; Li, Q.; Wu, Q.; Cheng, G. J.; Yang, M.; Sun, Y. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells. Anal. Chem. 2016, 88, 1979-1983. (20) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins S.; Futreal, P. A. Mutations of the BRAF Gene in Human Cancer. Nature 2002, 417, 949-954. (21) Dankort, D.; Curley, D. P.; Cartlidge, R. A.; Nelson, B.; Karnezis, A. N.; Damsky, W. E., Jr.; You, M. J.; DePinho, R. A.; McMahon, M.; Bosenberg, M. BrafV600E Cooperates with Pten Loss to Induce Metastatic Melanoma. Nat. Genet. 2009, 41, 544-552. (22) Das, J.; Ivanov, I.; Sargent, E. H.; Kelley, S. O. DNA Clutch Probes for Circulating Tumor DNA Analysis. J. Am. Chem. Soc. 2016, 138, 11009-11016. (23) Zhu, G.; Ye, X.; Dong, Z.; Lu, Y. C.; Sun, Y.; Liu, Y.; McCormack, R.; Gu, Y.; Liu, X. Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA From Patients with Advanced Non–Small Cell Lung Cancer. J. Mol. Diagn. 2015, 17, 265-272. (24) Chiou, C. C.; Luo, J. D.; Chen, T. L. Single-Tube Reaction Using Peptide Nucleic Acid as both PCR Clamp and Sensor Probe for the Detection of Rare Mutations. Nat. Protoc. 2006, 1, 26042612. (25) Amicarelli, G.; Shehi, E.; Makrigiorgos, G. M.; Adlerstein, D. FLAG Assay as a Novel Method for Real-Time Signal Generation During PCR: Application to Detection and Genotyping of KRAS Codon 12 Mutations. Nucleic Acids Res. 2007, 35, e131. (26) Hindson, B. J.; Ness, K. D.; Masquelier, D. A.; Belgrader, P.; Heredia, N. J.; Makarewicz, A. J.; Bright, I. J.; Lucero, M. Y.; Hiddessen, A. L.; Legler, T. C.; Kitano, T. K.; Hodel, M. R.; Petersen, J. F.; Wyatt, P. W.; Steenblock, E. R.; Shah, P. H.; Bousse, L. J.; Troup, C. B.; Mellen, J. C.; Wittmann, D. K.; Erndt, N. G.; Cauley, T. H.; Koehler, R. T.; So, A. P.; Dube, S.; Rose, K. A.; Montesclaros, L.; Wang, S.; Stumbo, D. P.; Hodges, S. P.; Romine, S.; Milanovich, F. P.; White, H. E.; Regan, J. F.; Karlin-Neumann, G. A.; Hindson, C. M.; Saxonov, S.; Colston, B. W. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Anal. Chem. 2011, 83, 8604-8610. (27) Miotke, L.; Lau, B. T.; Rumma, R. T.; Ji, H. P. High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR. Anal. Chem. 2014, 86, 2618-2624. (28) Tian, H.; Sun, Y.; Liu, C.; Duan, X.; Tang, W.; Li, Z. Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction. Anal. Chem. 2016, 88, 11384-11389. (29) Hu, Y.; Jia, H.; Wang, Y.; Cheng, Y.; Li, Z. Sensitive Quantification of Messenger RNA with a Real-Time Ligase Chain Reaction by Using a Ribonucleotide-Modified DNA Probe. Chem. Commun. 2014, 50, 13093-13095. (30) Deng, R.; Zhang, K.; Sun, Y.; Ren, X.; Li, J. Highly Specific Imaging of mRNA in Single Cells by Target RNA-Initiated Rolling Circle Amplification. Chem. Sci. 2017, 8, 3668-3675. (31) Nakamura, A.; Arita, T.; Tsuchiya, S.; Donelan, J.; Chouitar, J.; Carideo, E.; Galvin, K.; Okaniwa, M.; Ishikawa, T.; Yoshida, S. Antitumor Activity of the Selective Pan-RAF Inhibitor TAK-632 in BRAF Inhibitor-Resistant Melanoma. Cancer Res. 2013, 73, 70437055. (32) Leboeuf, R.; Baumgartner, J. E.; Benezra, M.; Malaguarnera, R.; Solit, D.; Pratilas, C. A.; Rosen, N.; Knauf, J. A.; Fagin, J. A. BRAFV600E Mutation Is Associated with Preferential Sensitivity to

ACS Paragon Plus Environment

ACS Sensors 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

Mitogen-Activated Protein Kinase Kinase Inhibition in Thyroid Cancer Cell Lines. J. Clin. Endocrinol. Metab. 2008, 93, 2194-2201. (33) Schwarzenbach, H.; Hoon, D. S.; Pantel, K. Cell-Free Nucleic Acids as Biomarkers in Cancer Patients. Nat. Rev. Cancer 2011, 11, 426-437. (34) Miura, N.; Nakamura, H.; Sato, R.; Tsukamoto, T.; Harada, T.; Takahashi, S.; Adachi, Y.; Shomori, K.; Sano, A.; Kishimoto, Y.; Ito, H.; Hasegawa, J.; Shiota, G. Clinical Usefulness of Serum Telomerase Reverse Transcriptase (hTERT) mRNA and Epidermal Growth Factor Receptor (EGFR) mRNA as a Novel Tumor Marker for Lung Cancer. Cancer Sci. 2006, 97, 1366-1373.

Page 8 of 14

(35) O’driscoll, L.; Kenny, E.; Mehta J. P.; Doolan, P.; Joyce, H.; Gammell, P.; Hill, A.; Clynes, M. Feasibility and Relevance of Global Expression Profiling of Gene Transcripts in Serum From Breast Cancer Patients Using Whole Genome Microarrays and Quantitative RT-PCR. Cancer Genom. Proteom. 2008, 5, 95-104. (36) Slonim, D. K.; Koide, K.; Johnson, K. L.; Tantravahi, U.; Cowan, J. M.; Jarrah, Z.; Bianchi, D. W. Functional Genomic Analysis of Amniotic Fluid Cell-Free mRNA Suggests that Oxidative Stress is Significant in Down Syndrome Fetuses Proc. Natl. Acad. Sci. USA 2009, 106, 9425-9429.

ACS Paragon Plus Environment

Page 9 of 14 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

ACS Sensors

for TOC only

ACS Paragon Plus Environment

ACS Sensors 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

153x269mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 10 of 14

Page 11 of 14 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

ACS Sensors

80x76mm (300 x 300 DPI)

ACS Paragon Plus Environment

ACS Sensors 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

66x51mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 12 of 14

Page 13 of 14 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

ACS Sensors

33x13mm (300 x 300 DPI)

ACS Paragon Plus Environment

ACS Sensors 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

64x49mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 14 of 14