{'fraction_non_alphanumeric': 0.047193833060099756, 'fraction_numerical': 0.0374620670410548, 'mean_word_length': 4.768611670020121, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction.


Introduction
Lung cancer is the leading cause of cancer death and accounts for 18.0% of the total cancer deaths. The survival of lung cancer patients in 5 years aer diagnosis is only 10%-20%. 1 Imaging and tissue biopsy are the standard methods for lung cancer diagnosis, but they suffer from ionizing radiation exposure and invasiveness. 2 Traditional tumor diagnostic markers include carcinoembryonic antigen (CEA) and carcinoma antigen 125 (CA125), and they usually exhibit low sensitivity. 3 Due to their simpler structure and less post-processing than DNA and protein, microRNAs (miRNAs) can function as potential noninvasive biomarkers for early diagnosis and prognosis of lung cancer. [4][5][6][7][8][9] miRNAs are short endogenous non-coding RNAs with a length of $22 nucleotides (nt). miRNAs can regulate the expression of specic genes by binding to complementary regions of messenger RNA (mRNA), and the resulting duplex may inhibit translation by either enhancing the degradation or blocking the initiation of mRNA. 10 More than 60% of human protein-coding genes harbor the predicted miRNA target sites. miRNAs play crucial regulatory roles in animal development, cell differentiation, apoptosis, proliferation and immunity, [11][12][13] while dysregulations of miRNAs are frequently observed in a variety of cancers, 14 [15][16][17] and the upregulation of miR-21 in lung cancer. [18][19][20] Consequently, the detection of multiple miRNAs is essential for early diagnosis of lung cancer.
Due to the properties of small size, low abundance in real biological tissues and short survival time in vitro, sensitive detection of miRNAs is very challenging. 21 Traditional methods include polymerase chain reaction (PCR), 22 microarray analysis, 23 and northern blotting. 24 PCR-based methods exhibit high sensitivity, but they involve the specially designed primers and precise temperature control. 22 Although microarray analysis is suitable for multiple miRNA assay, it suffers from high cost, narrow dynamic range, low sensitivity, and poor repeatability. 23 Northern blot is a common method for miRNA assay, but it suffers from long analysis time, low sensitivity, and large sample consumption. 24 Alternatively, a series of new methods have been developed for miRNA assay based on electrochemical, 25 colorimetric, 26 Raman spectrum, 27,28 and uorescence measurements. [29][30][31] However, their sensitivities are relatively low without the involvement of signal amplication. [25][26][27][28][29][30][31] To improve the sensitivity, several signal amplication strategies such as hybridization chain reaction, 32,33 strand displacement amplication, 34,35 rolling circle amplication, 36 and DNA hydrogelation-based signal amplication 37 have now been introduced for the detection of a single type of miRNA. [33][34][35][36][37] Notably, one disease may correlate with the mis-expression of different types of miRNAs, 38 and the combination of multiple miRNAs may greatly improve the diagnostic accuracy. 39,40 Therefore, new methods with the capability of sensitive detection of multiple miRNAs are highly required.
Herein, we demonstrate the integration of multicolor uorophore-encoded cascade signal amplication with singlemolecule detection for simultaneous detection of miRNAs in lung cancer tissues. Single-molecule detection has gained overwhelming attention due to the high signal-to-noise ratio, low sample consumption, and ultrahigh sensitivity. [41][42][43][44][45][46] We used two lung cancer biomarkers miR-155 and miR-21 as the targets. The presence of miR-155 and miR-21 can initiate three consecutive amplication reactions (i.e., cyclic strand displacement amplication (SDA), APE1-assisted cyclic cleavage, and rolling circle amplication (RCA)), converting extremely low abundant target miRNAs to abundant Cy5/Cy3 uorophore-encoded DNA products whose digestion releases larger numbers of uorescent molecules. The subsequent single-molecule detection enables the identication of miR-155 with Cy5 and miR-21 with Cy3. Due to the high efficiency of the cascade signal amplication and high signal-to-noise ratio of single-molecule detection, this method can measure miRNAs with a detection limit of 25.7 aM for miR-155 and 45.7 aM for miR-21. Moreover, this assay can simultaneously quantify the expression levels of miR-21 and miR-155 in living cells and lung cancer tissues at the single-molecule level, and it can distinguish non-small cell lung cancer (NSCLC) patients from healthy persons as well.
Scheme 1 Principle of simultaneous detection of multiple miRNAs based on the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection.

Results and discussion
Principle of multiple miRNA assay The design for simultaneous detection of multiple miRNAs is shown in Scheme 1. We designed a linear template, a biotinylated AP probe, and a circular template for each miRNA. The 3 0 ends of each linear template and each AP probe are modied with NH 2 to prevent nonspecic amplication. 47 The linear template-155 consists of a miR-155-binding region, a recognition site for the Nt.BstNBI nicking enzyme, and an amplication region for miR-155-induced cyclic SDA. The linear template-21 consists of a miR-21-binding region, a recognition site for Nt.BstNBI, and an amplication region for miR-21induced cyclic SDA. The biotinylated AP probe-155 consists of a binding region for trigger-155 generated from the rst cyclic SDA and an AP site as the recognition domain for APE1-assisted cyclic cleavage. The biotinylated AP probe-21 consists of a binding region for trigger-21 generated from the rst cyclic SDA and an AP site. The circular template-155 consists of a special binding domain for primer-155 released from the second APE1-assisted cyclic cleavage and an amplication domain for the RCA reaction. The circular template-21 consists of a special binding domain for primer-21 released from the second APE1-assisted cyclic cleavage and an amplication domain for the RCA reaction.
This assay contains the following steps: (1) target miRNAinduced cyclic SDA, (2) APE1-assisted cyclic cleavage, (3) RCA reaction, (4) magnetic separation and the cleavage of amplied products by Exo I/Exo III, and (5) single-molecule detection. In the presence of miR-155 (red color, Scheme 1), the binding of linear template-155 to miR-155 forms a partial duplex, and subsequently miR-155 can be extended along linear template-155 to generate a dsDNA in the presence of Vent (exo-) DNA polymerase and dNTPs. The resultant dsDNA can be cut by Nt.BstNBI at the cleavage recognition domain, generating trigger-155 and a new replication site for DNA polymerase. Consequently, abundant trigger-155 can be produced through repeated extension, cleavage and trigger generation (the rst signal amplication). The resultant trigger-155 can bind to AP probe-155 to obtain the partial dsDNA with an AP site. Upon the addition of APE1, the partial dsDNA is cleaved at the AP site, generating 5 0 biotinylated primer-155 and simultaneously releasing trigger-155. The free trigger-155 can further hybridize with new AP probe-155 repeatedly, initiating the APE1-assisted cyclic cleavage of biotinylated AP probe-155 through repeated hybridization, cleavage and dissociation, generating abundant 5 0 biotinylated primer-155 (the second signal amplication). In the meantime, shrimp alkaline phosphatase (rSAP) is added into the reaction solution to hydrolyse the excess dNTPs from the rst step. Subsequently, the resultant primer-155 binds to circular template-155 to initiate the RCA reaction in the presence of phi29 polymerase, dATP, dTTP, Cy5-dCTP and Cy3-dGTP. The circular template-155 containing only three types of bases (i.e., A, T, and G) can generate the RCA product with only three types of bases (i.e., T, A, and C), and the introduction of Cy5-modied dCTP enables the incorporation of abundant Cy5 uorescent molecules into the RCA product (the third signal amplication). Notably, target miR-155 induces the rst cyclic SDA reaction to yield many trigger-155 which can subsequently initiate the second APE1-assisted cyclic cleavage, producing more 5 0 biotinylated primer-155. Each resultant 5 0 biotinylated primer-155 may produce a biotin-/multiple Cy5labeled ssDNA through the third RCA reaction, resulting in the generation of abundant biotin-/multiple Cy5-labeled ssDNAs. The ssDNA products can be captured by streptavidincoated magnetic beads (MBs) and separated from the reaction solution through magnetic separation. Then the product is digested into single nucleotides by Exo I/Exo III to release large numbers of Cy5 uorescent molecules, which can be simply measured by single-molecule detection.
When miR-21 is present (green color, Scheme 1), it can partially bind to linear template-21 to initiate the cyclic SDA reaction, producing abundant trigger-21. The resultant trigger-21 binds to AP probe-21 to initiate the APE1-assisted cyclic cleavage, generating large numbers of 5 0 biotinylated primer-21. Subsequently, the resultant primer-21 can react with circular template-21 to initiate the RCA reaction, and the introduction of Cy3-modied dGTP enables the incorporation of abundant Cy3 uorescent molecules into the RCA product. When miR-155 and miR-21 coexist, they induce three consecutive amplication events (i.e., target miRNA-induced cyclic SDA, APE1-assisted cyclic cleavage, and RCA reaction), generating abundant biotin-/multiple Cy5-labeled ssDNA products for miR-155 and biotin-/multiple Cy3-labeled ssDNA products for miR-21. The subsequent digestion of amplication products by Exo I/Exo III releases large numbers of uorescent molecules which can be simultaneously quantied by single-molecule detection, with Cy5 indicating miR-155 and Cy3 indicating miR-21. However, in the absence of miRNAs, three consecutive amplication events cannot occur, and neither Cy5 nor Cy3 signals can be observed.

Validation of the assay
We used 14% native polyacrylamide gel electrophoresis (PAGE) to analyze the products of the target miRNA-induced cyclic SDA reaction (Fig. 1A). Only a 23 nt band is observed for miR-155 alone ( To verify if the binding of the triggers to the AP probes leads to the APE1-assisted cyclic cleavage, we measured the melting curves of the reaction products ( Fig. 1B and C). For the trigger-155/AP probe-155 duplex, the melting temperature of the product is 36.5 C aer APE1 treatment (Fig. 1B, red line), much lower than that before APE1 treatment (63.5 C; Fig. 1B, black line), indicating the occurrence of the APE1-assisted cyclic cleavage reaction. Similarly, for the trigger-21/AP probe-21 duplex, the melting temperature of the product is 33.5 C aer APE1 treatment (Fig. 1C, olive line), much lower than that before APE1 treatment (61.5; Fig. 1C, blue line).
We used agarose gel electrophoresis to conrm the target miRNA-induced RCA reaction (Fig. 1D). No amplication is observed when miR-21 and miR-155 are absent (Fig. 1D, lanes 4-6). However in the presence of miR-155, the bands of ampli-ed DNAs are observed (Fig. 1D, lane 1), suggesting the occurrence of three consecutive amplication events including the miR-155-induced cyclic SDA, APE1-assisted cyclic cleavage, and the subsequent RCA reaction. Similarly, the bands of amplied DNAs are observed in response to miR-21 (Fig. 1D,  lane 2), suggesting the occurrence of miR-21-induced three consecutive amplication events. Moreover, the bands of amplied DNAs are observed when both miR-155 and miR-21 are present (Fig. 1D, lane 3).
We further used uorescence measurement to demonstrate the target miRNA-induced RCA and the incorporation of uorescent molecules into RCA products ( Fig. 1E and F). When miR-155 is present, a distinct Cy5 uorescence signal is detected (Fig. 1E, red line). Similarly, a distinct Cy3 uorescence signal is detected when miR-21 is present (Fig. 1F, olive line). However, no distinct uorescence signal can be detected in the absence of miR-155 (Fig. 1E, black line) and miR-21 (Fig. 1F, blue line). The results demonstrate that the miRNA-induced RCA reaction can only be triggered by targets miR-155 and miR-21 in the presence of specic linear templates, AP probes, and circular templates.
Under the optimal conditions ( Fig. S1-S4 †), we measured uorescence emission spectra generated by different concentrations of target miRNAs. For miR-155 assay, the Cy5 uorescence signal gradually enhances with the increasing concentration of miR-155 from 0 to 3 Â 10 À8 M (Fig. S5A †), and the uorescence intensity (F) shows a linear correlation with the logarithm of miR-155 concentration (C) in the range of 1 Â 10 À15 -1 Â 10 À10 M (Fig. S5B †). The correlation equation is F ¼ 402 447 + 25 902 log 10 C (R 2 ¼ 0.9920). The limit of detection (LOD) is 9.83 Â 10 À16 M by extrapolating the concentration from the signal equal to the mean control signal plus three standard deviations of the control signal. For miR-21 assay, the Cy3 uorescence signal improves with the increasing concentration of miR-21 from 0 to 3 Â 10 À8 M (Fig. S5C †), and the uorescence intensity (F) shows a linear correlation with the logarithm of miR-21 concentration (C) in the range of 1 Â 10 À14 -1 Â 10 À10 M (Fig. S5D †). The correlation equation is F ¼ 326 419 + 21 874 log 10 C (R 2 ¼ 0.9948). The LOD is evaluated to be 5.01 Â 10 À15 M.

Single-molecule/particle detection
We employed single-molecule/particle imaging to measure different target miRNA-induced uorescence encoding. Aer the magnetic beads were incubated with the RCA reaction products, the product-coupled magnetic beads displayed distinct uorescence images in the red and green channels under the excitation of 640 nm and 561-nm lasers, respectively. When miR-155 is present alone, only the Cy5 signal can be detected (Fig. 2B(a), red color) and no Cy3 signal is observed (Fig. 2C(a)). When miR-21 is present alone, only the Cy3 signal can be detected (Fig. 2C(e), green color), and no Cy5 signal can be detected (Fig. 2B(e)). When both miR-155 and miR-21 are present, both Cy5 ( Fig. 2B(b-d and f), red color) and Cy3 ( Fig. 2C(b-d and f), green color) signals are simultaneously detected. Moreover, the colocalization of Cy5 and Cy3 uorescence signals leads to the appearance of yellow color signals ( Fig. 2A(b-d and f)).
For the optical coding, two different RCA reaction products with distinctively different uorescence emission spectra (i.e., Cy5 and Cy3) are used to modify the magnetic beads. Five types of color-encoded magnetic beads are identied at the singleparticle level. There are red ( Fig. 2A(a)), yellow ( Fig. 2A(c and  f)), and green color alone ( Fig. 2A(e)), a red region appearing in the periphery of a yellow region ( Fig. 2A(b)), and a green region appearing in the periphery of a yellow region ( Fig. 2A(d)). The size of the region around each spot is primarily associated with the concentration of target miRNAs. Notably, the increase of miRNA concentration can induce the formation of larger size product-coupled magnetic beads, generating red/green monomers with tunable size for each spot. The red-green heterodimers display yellow colocalized signals. Thus, a colorimetric signal change at the single-particle level can be quantitatively analyzed with the color proling method.
Multi-signal magnetic beads can be rationally designed and exploited for the simultaneous detection of miRNAs. We prepared a series of articial mixtures by mixing miR-155 and miR-21. Fig. 2B(a-e) shows that the increase of miR-155 concentration can induce the formation of larger size productcoupled magnetic beads in the red channel. As expected, the largest size is detected in response to 30 nM miR-155 ( Fig. 2B(a), red color), whose size is consistent with the size in response to 30 nM miR-155 + 30 nM miR-21 ( Fig. 2B(f), red color). These results indicate that this assay is capable of detecting miR-155 even in the presence of miR-21. Similarly, in the presence of increasing concentration of miR-21, product-coupled magnetic beads exhibit increased size in the green channel ( Fig. 2C(a-e)). The largest size is detected in the presence of 30 nM miR-21 ( Fig. 2C(e), green color), whose size is consistent with the size in the presence of 30 nM miR-155 + 30 nM miR-21 ( Fig. 2C(f), green color). Thus, this method is suitable for the visualization of miRNA concentration changes.
The distribution of the uorescence intensity of the spot was further analyzed. Upon the addition of increased concentration of miR-155 (0-30 nM), the Cy5 uorescence intensity is significantly enhanced (Fig. 2D(a-e)). When the miR-155 concentration is 30 nM, the uorescence intensity reaches the highest value ( Fig. 2D(a)). The mixture of miR-155 and miR-21 gives rise to an identical Cy5 intensity (Fig. 2D(f)). When an increased concentration of miR-21 is added to the mixture, the Cy3 intensity is gradually enhanced (Fig. 2E(a-e)), and the highest value is obtained at a concentration of 30 nM (Fig. 2E(e)). There is no signicant difference between miR-21 alone and the mixture (Fig. 2E(e and f)). The results demonstrate that this method can be used for monitoring the concentration changes of both miR-21 and miR-155 at the single-particle level.
We further employed this method to measure different concentrations of miR-155 and miR-21. The numbers of Cy5 (Fig. S6A †) and Cy3 uorescence signals (Fig. S6B †) enhance with the increasing concentrations of miR-155 and miR-21, respectively. Moreover, the Cy5 counts (Fig. 4A) and Cy3 counts (Fig. 4B) improve with the increasing concentration of miR-155 and miR-21 in the range from 0 M to 3 Â 10 À8 M, respectively, and the number of uorescent molecules (N) exhibits a linear correlation with the logarithm of miRNA concentration (C) in the range of 1 Â 10 À16 -1 Â 10 À11 M (inset of Fig. 4A and B). For miR-155 assay, the correlation equation is N ¼ 738.6 + 43.66 log 10 C (R 2 ¼ 0.9911), and the LOD is measured to be 2.57 Â 10 À17 M. The sensitivity of this method has been improved by 38-fold compared with that of ensemble uorescence measurement (Fig. S5B †). For miR-21 assay, the correlation equation is N ¼ 648.6 + 38.37 log 10 C (R 2 ¼ 0.9909), and the LOD is measured to be 4.57 Â 10 À17 M. The sensitivity of this method has been improved by 110-fold compared with that of ensemble uorescence measurement (Fig. S5D †). Notably, the proposed method enables simultaneous and sensitive detection of miR-155 and miR-21 with a linear range of 5 orders of magnitude, superior to the reported methods for miRNA assays (Table S1 †). The sensitivity of this method has improved by as much as 4 orders of magnitude compared with that of quantum dots and graphene oxide nano-photon switchbased uorescent assay, 48 2 orders of magnitude compared with the peptide nucleic acid-based electrochemical biosensor, 49 and 2 orders of magnitude compared with the bi-directional DNA walking machine-based electrochemiluminescent biosensor. 50

Measurement of miRNAs in clinical tissue samples
Previous research studies demonstrate the high expression levels of miR-21 and miR-155 in patients with non-small cell lung cancer (NSCLC). 53,54 To conrm the proof-of-concept of this assay for real clinical sample analysis, we analyzed human tissue samples from twelve diagnosed NSCLC patients and six healthy persons. As shown in Fig. 7, the miR-155 concentration in NSCLC patients is signicantly higher than that in healthy persons (t test, P < 0.0001), with a median concentration of 18.5 pM for NSCLC patients and 1.42 pM for healthy persons. Moreover, the miR-21 concentration in NSCLC patients is signicantly higher than that in healthy persons (t test, P < 0.0001), with a median concentration of 94.2 pM for NSCLC patients and 4.08 pM for healthy persons. We further validated the results of the proposed method using qRT-PCR (Fig. 7, dark gray column). The results obtained by the proposed method are in good agreement with those obtained by qRT-PCR for each sample (Fig. 7). The results suggest that miR-155 and miR-21 can function as tumor biomarkers for early diagnosis of NSCLC.

Conclusions
In summary, we demonstrate the integration of multicolor uorophore-encoded cascade signal amplication with singlemolecule detection for the simultaneous measurement of miRNAs in lung cancer tissue samples. This assay involves three consecutive amplication reactions which enable the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 uorophore-encoded DNA products which can release large numbers of uorescent molecules for the generation of amplied signals. Unlike the reported methods for miRNA assay with the requirement of carefully designed probes (e.g., hairpin probes labelled with different-color quantum dots, 48 signal probes labelled with Cy3/Cy5, 55 hairpin probes labelled with ferrocene/methylene blue, 49 and the magnetic DNA nanosphere-encoded probes labelled with ferrocene/methylene blue 56 ), this assay enables the simultaneous detection of miRNAs without the need for either miRNA purication or multiple uorescent-labelled probes. Due to the high efficiency of the cascade signal amplication and high signal-to-noise ratio of single-molecule detection, this assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and lung cancer tissues, and it can distinguish the expression of miRNAs between NSCLC patients and healthy persons. We further validate the results of the proposed method using qRT-PCR. Importantly, the proposed method can be extended to detect other cancerrelated miRNAs by simply changing the sequence of linear templates, holding great potential in multiplexed detection and clinical diagnosis.

Chemicals and materials
All oligonucleotides (Table 1)

RCA reaction
The RCA reaction was performed in 25 mL of reaction mixture containing the above reaction solution, 96 nM circular template-155, 96 nM circular template-21, 240 mM dATP, 240 mM dTTP, 8 mM Cy5-dCTP, 8 mM Cy3-dGTP, 5 U of phi29 DNA polymerase, and 0.5Â phi29 reaction buffer at 30 C for 1.5 h in the dark, and the reaction was terminated by incubation at 65 C for 10 min.

Conjugation of RCA products with the streptavidin-coated MBs and exonuclease digestion reaction
Prior to the reaction, 5 mL of streptavidin-coated MB solution (10 mg mL À1 ) was washed twice with 2Â binding and washing (B&W) buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 2 M NaCl) and resuspended in 2Â B&W buffer with a nal concentration of 2 mg mL À1 . Then 2 mg mL À1 MB solution was mixed with biotinylated RCA products, followed by incubation in the dark for 15 min on a roller mixer at room temperature. The mixture was then washed three times by magnetic separation using 1Â B&W buffer to remove the excess Cy5-dCTP and Cy3-dGTP, and the single-stranded DNA-MB conjugates (MB-ssDNAs) were resuspended in 1Â NEBuffer 1. The exonuclease digestion reaction was performed in 20 mL of reaction mixture containing MB-ssDNAs, 10 U of Exo I, 10 U of Exo III, 1Â Exo I reaction buffer, and 1Â NEBuffer 1 at 37 C for 30 min. Finally, the streptavidin-coated MBs were separated by magnetic separation for 3 min in the dark, and the supernatant solution was subjected to subsequent detection.

Gel electrophoresis and uorescence measurement
The products of the target miRNA-induced cyclic SDA reaction were analyzed by 14% nondenaturing polyacrylamide gel electrophoresis (PAGE) in TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, and 0.2 mM EDTA) at a constant voltage of 110 V for 50 min at room temperature. Then the gel was stained with SYBR gold and analyzed by using a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA). The RCA reaction products stained with SYBR gold were analyzed by 1% agarose gel electrophoresis in TAE buffer (40 mM Tris-acetic acid and 2 mM EDTA) at a constant voltage of 110 V for 60 min. 20 mL of the reaction products were diluted to a nal volume of 80 mL with ultrapure water, and subsequently subjected to uorescence emission spectra measurement by using an FLS1000 (Edinburgh Instruments, UK). Cy5 uorescence was measured at an excitation wavelength of 635 nm, with the uorescence intensity at 668 nm being used for the quantitative analysis of miR-155. Cy3 uorescence was measured at an excitation wavelength of 532 nm, with the uorescence intensity at 568 nm being used for the quantitative analysis of miR-21.

Single-molecule detection and data analysis
For single-molecule measurement, the reaction products were diluted 10 000-fold with 1Â imaging buffer (3 mM MgCl 2 , 100 mM Tris-HCl, pH 8.0, and 10 mM (NH 4 ) 2 SO 4 ). 10 mL of samples were spread on a glass coverslip for imaging. The images were acquired by total internal reection uorescence (TIRF) microscopy (Nikon, Ti-E, Japan). 640 nm and 561 nm lasers were used to excite Cy5 and Cy3 uorescent molecules, respectively. The photons of Cy5 and Cy3 were collected by an oil immersion 100Â objective (Nikon, Japan), and were split into the Cy5 channel (661.5-690.5 nm lter) and Cy3 channel (573-613 nm lter) by a dichroic mirror, and were imaged onto a digital CMSO EMCCD camera (Hamamatsu Photonics K. K., Japan) with an exposure time of 500 ms. For data analysis, regions of interest of 600 Â 600 pixels were selected for Cy5 and Cy3 molecule counting by using Image J soware (NIH, Bethesda, MD, USA). The numbers of Cy5 and Cy3 were the average of ten frames, respectively.

Real sample analysis
MDA-MB-231 cells, MCF-7 cells, A549 cells, HeLa cells, and HL-7702 cells were cultured with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Invitrogen, USA) in Dulbecco's modied Eagle's medium (DMEM). The cells were cultured at 37 C in a humidied atmosphere containing 5% CO 2 . The number of cells was counted by using a Countstar cell counter. The RNA extracts were prepared by using the miRNeasy Mini Kit according to the manufacturer's protocol. The extraction of total RNA from lung tissues of NSCLC patients and healthy persons embedded in paraffin was performed by using the miRNeasy FFPE kit according to the manufacturer's protocol. The concentration of total RNA was determined by using a NanoDrop 2000c spectrophotometer (Thermo Scientic, Wilmington, Delaware, USA).