A label-free fluorescence assay for microRNAs based on linear enzymatic signal amplification

Abstract

MicroRNAs (miRNAs) are regulators in various physiological and pathological processes with significant potential for disease diagnosis and therapeutic monitoring. However, their quantification remains a challenge due to their high degradability, short sequence length, and naturally low abundance in sample specimen. Herein, we report a fluorescence assay based on an innovative linear enzyme-assisted isothermal signal amplification strategy and label free fluorescence readout by means of an RNA-intercalating reagent. Poly(A) polymerase-catalyzed polyadenylation of target miRNA retained on the solid surface of magnetic beads resulted in a poly(A) tail of ∼150 bp added to the 3′ end of miRNA. The elongation of the miRNA sequence enables robust fluorescence staining with SYBR Green II, thereby allowing highly sensitive fluorescence detection. The assay proposed allows quantification of target miRNAs with a detection limit of 66.4 fM and a linear calibration relating fluorescence intensity to miRNA concentration (R² = 0.998). Its applicability was demonstrated by quantifying target miRNAs in cellular samples. With its high sensitivity and accuracy, the assay successfully detected paclitaxel-induced downregulation of miRNA-146a-5p in MDA-MB-231 breast cancer cells. Collectively, these results highlight the potential of this assay for quantitative miRNA analysis in biomedical and clinical applications.

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1. Introduction

MicroRNAs (miRNAs) are short, single-stranded, non-coding RNA molecules, typically 20–25 nucleotides in length. They modulate gene function across a variety of biological processes.^1,2 They bind to complementary sequences in target transcripts' 3′ untranslated regions, which causes transcript degradation, post-transcriptional gene expression, and suppression of protein synthesis.³ Recent research has identified miRNA interactions with alternative genomic regions, such as the 5′ UTR, coding sequences, and promoter regions, thereby expanding their functional range to encompass both gene silencing and activation mechanisms.^4,5 These interactions allow miRNAs to influence essential cellular mechanisms such as growth, isolation, cell death, and contribute to the development of numerous clinical disorders.^6,7 Increasing research highlights the crucial function of miRNAs in oncogenic pathways, e.g., in the initiation and progression of breast cancer.^8–11 Quantification of miRNAs in biological samples remains a challenge due to their short length, vulnerability to degradation, and often trace levels in biofluids.^12–14 Over the past few years, analytical methods based on diverse techniques have been reported, including quantitative reverse transcription PCR (RT-qPCR),¹⁵ microarray analysis,¹⁶ next-generation sequencing (NSG),¹⁷ electrochemical sensors,¹⁸ and fluorescence-based assays.^19–21 RT-qPCR in particular offers high sensitivity and specificity but suffers from intrinsic drawbacks when handling very short targets namely, low primer-template melting temperatures that hinder hybridization, off-target priming from single-base mismatches, and risks of primer extension artifacts or sample contamination.^22,23 In response, recent work has focused on enzyme-assisted isothermal amplification strategies coupled with fluorescence readout, including rolling circle amplification, loop-mediated isothermal amplification, and duplex-specific nuclease signal amplification. Most isothermal amplification-based fluorescence assays reported previously require fluorescently labeled DNA probes, which cause problems associated with high background signals, limited shelf lifetime of the probes, and certainly assay costs. Also importantly, in these assays, linear calibration curves were constructed between fluorescence intensity and the logarithm of analyte concentrations (instead of analyte concentrations), compromising assay accuracy due to its mathematical basis.²⁴ In recent years, the development of PCR-free methods for miRNA quantitative assay has been heavily reported, and reviews on this subject can be found in the literature.^25–27

In this work, we studied a novel isothermal signal amplification strategy for miRNA quantification. The strategy was based on poly(A) polyadenylation of target miRNA for linear signal amplification. The elongation of target miRNA enables effective fluorescence staining with an RNA-intercalating reagent, i.e., SYBR Green II. This strategy enabled the development of a label-free fluorescence assay for quantifying target miRNAs in biological samples. Analytical figures of merit were assessed for the method. Its application in quantitative analysis of cellular samples was demonstrated with miRNA-146a-5p as the model analyte.

2. Materials and methods

2.1. Reagents and samples

Chemicals including sodium chloride (NaCl), magnesium chloride (MgCl₂), hydroxymethyl aminomethane (Tris-base), ethylenediaminetetraacetic acid (EDTA), boric acid, and Triton X-100 were acquired from Thermo Scientific. RNase-free water utilized in the experiments was obtained from Invitrogen. Streptavidin-coated magnetic beads (1 µm in diameter) were purchased from Sigma-Aldrich. Oligonucleotide sequences for target miRNAs and the complementary ssDNA probes are listed in Table 1. Qiagen supplied E. coli poly(A) polymerase, ATP, 10× poly(A) polymerase reaction buffer, and RNase inhibitor. Electrophoresis reagents including 2% agarose E-Gel and SYBR Green II RNA gel stain (10 000× concentrate in DMSO) were obtained from Thermo Fisher. Bio-Rad provided 30% acrylamide/bisacrylamide solution (29 : 1, v/v), 10% ammonium persulfate (APS), and tetramethyl ethylenediamine (TEMED). Centrifuge tubes and pipette tips were autoclaved for sterilization, and all buffers were prepared using RNase-free water to ensure miRNA stability. The 1× poly(A) polymerase reaction buffer contained 50 mM Tris–HCl, 250 mM NaCl, and 10 mM MgCl₂ (pH 8.1 at 25 °C). The binding/washing buffer comprising 10 mM Tris buffer (pH 7.5), 2 M NaCl, 1 mM EDTA, and 0.0005% (w/v) Triton X-100. SYBR Green II working solution was prepared by mixing 1 µL of stock solution with 49.9 mL of TBE buffer (89 mM Tris base, 89 mM boric acid, 1 mM EDTA, pH 8) to achieve a 1 : 50 000 dilution. HEK-293 and MDA-MB-231 cells were obtained from ATCC and maintained in the laboratory following the vendor's protocols.

Table 1 miRNAs and their biotinylated ssDNA probe sequences used in this study

miRNAs	Mature miRNA sequences	Complementary ssDNA probes
hsa-miR-146a-5p	5′-UGA GAA CUG AAU UCC AUG GGU U-3′	5′-TGG AAT TCA GTT CTC A-TEG-biotin-3′
hsa-miR-10a-5p	5′-UAC CCU GUA GAU CCG AAU UUG UG-3′	5′-TTC GGA TCT ACA GGG TA-TEG-biotin-3′

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2.2. Preparation of magnetic beads-ssDNA probe conjugates

A 100 µL aliquot of streptavidin-coated magnetic beads (1 µm in diameter) was transferred into a sterile microcentrifuge tube and subjected to two washes with 500 µL of binding/washing buffer (10 mM Tris–HCl, 1 mM EDTA, 2 M NaCl, pH 8.0). After each washing, the beads were collected by centrifugation, the supernatant was carefully removed, and the pellet was resuspended in fresh buffer. To the suspension, 10 µL of the biotinylated ssDNA probe (final concentration 1.0 mM) was added. The mixture was incubated at 37 °C for 30 minutes to facilitate binding between the biotin-labelled probes and streptavidin-coated beads. After incubation, the magnetic beads were collected by centrifugation, the supernatant was discarded, and the beads were washed 3× with binding/washing buffer to remove unbound probes. To block remaining streptavidin binding sites, the beads were incubated with 100 µM free biotin (prepared in binding/washing buffer) under slow orbital mixing for 30 min at ambient temperature.²⁸ After biotin blocking, the beads were collected by centrifugation, the supernatant was removed, and a final wash with binding/washing buffer was performed. The resulting ssDNA probe-magnetic bead conjugates were resuspended in 2.0 mL of binding/washing buffer and stored at 4 °C for subsequent use.

2.3. Fluorescence assay of miRNA

A sample solution (100 µL) was added to 200 µL of the MBs-ssDNA probe conjugate suspension prepared above. The mixture was incubated at 37 °C for ¹H on a shaker. After incubation, MBs were collected by centrifugation and were then washed once with the binding/washing buffer and once with 1× poly(A) polymerase reaction buffer. After supernatant removal, the polyadenylation reaction mixture was prepared by adding 1× poly(A) polymerase reaction buffer, adenosine triphosphate (ATP, 10 mM), poly(A) polymerase (5 U µL⁻¹), oligo (dT) (0.5 µM), and RNase inhibitor (2 U µL⁻¹), with RNase-free water added to achieve a final reaction volume of 100 µL. The reaction mixture was gently mixed and incubated at 37 °C for 2 hours to facilitate polyadenylation. Following incubation, magnetic beads were washed five times with 1× poly(A) polymerase reaction buffer to remove unbound components, including ATP and oligo(dT). Thermal denaturation (release) of the heteroduplexes immobilized on the magnetic beads was performed by heating 200 µL of RNase-free water at 95 °C for 5 minutes. The extended miRNA solution (100 µL) was mixed with 500 µL of SYBR Green II prepared in TBE buffer (89 mM Tris base, 89 mM boric acid, 1 mM EDTA, pH 8.0) and 100 µL of 0.0005% (w/v) Triton X-100. Fluorescence was measured at 450 nm/508 nm.

3. Results and discussion

3.1. Protocol design

Isothermal amplification-based fluorometric methods for miRNA quantification are sensitive and specific. However, most of these methods require fluorescently labeled DNA probes and have calibration curves of fluorescence intensity versus the logarithm of miRNA concentration. To overcome these disadvantages, we investigated a new assay strategy that involves poly(A) polymerase-catalyzed polyadenylation of target miRNA for signal amplification and fluorescence staining of extended miRNA with SYBR Green II, an RNA-intercalating dye for fluorescence readout. Unlike the enzymes previously reported for isothermal signal amplification in miRNA fluorescence assays such as duplex-specific nucleases, poly(A) polymerase demonstrates strong catalytic efficiency, creating an elongated product (i.e., extended miRNA). It was expected that this enzymatic reaction mechanism offers linear signal amplification. Also importantly, elongation of target miRNA allows effective fluorescence staining by using an RNA-intercalating dye. SYBR Green II is selected for its high RNA bound quantum yield (∼0.56) and stability in solution.²⁹Fig. 1 shows the workflow of the proposed assay strategy: (1) capture of target miRNA with the magnetic bead-ssDNA probe conjugate provides high specificity through sequence-selective hybridization and enables efficient separation of target-bound complexes from impurities; (2) poly(A) polymerase-catalyzed polyadenylation adds a polyadenine tail to the 3′ end of miRNA immobilized on magnetic beads; (3) fluorescence staining of extended miRNA released from magnetic beads by denaturing with SYBR Green II for fluorometric measurements.

Fig. 1 Workflow for the proposed quantitative assay of target miRNAs.

3.2. Poly(A) polymerase-catalyzed polyadenylation of miRNA on a solid surface versus free solution

Although polyadenylation of RNAs is well documented, no study has reported it on a solid surface. In this work, gel electrophoresis was performed to evaluate poly(A) tail extension for both conventional solution-phase and solid surface-based polyadenylation approaches. To ensure rigor, we performed the separation on both 2% agarose and 5% polyacrylamide gels. As shown in Fig. 2, polyadenylation in free solution resulted in an extended miRNA of ∼95 base pairs, while on the solid surface of magnetic beads an extended miRNA of ∼150 base pairs. Immobilization of target miRNA on magnetic beads improved catalytic efficiency of poly(A) polymerase, creating a significantly elongated product. This solid surface-based enzymatic reaction mechanism offers enhanced target specificity through spatial organization of the polyadenylation reaction. Also importantly, the extended poly(A) tail length achieved through bead-based immobilization enables more sensitive fluorescence detection using the RNA-intercalating dye. These results indicate that the magnetic bead-based polyadenylation strategy provides both improved target specificity and enhanced poly(A) tail extension efficiency relative to solution-phase protocols.

Fig. 2 Gel electrophoresis-based comparison of poly(A)-tail lengths from free solution versus solid surface polyadenylation: (A) analysis on a 2% agarose gel and (B) on a 5% polyacrylamide gel.

3.3. Fluorescence staining of extended miRNA with SYBR Green II

SYBR Green II is a well-known RNA-intercalating fluorescence dye. The dye is weakly fluorescent in solution due to non-radiative relaxation, but when bound (intercalated) into RNA base stacks, molecular motion is restricted, non-radiative decay is suppressed, and fluorescence emission is strongly enhanced. Theoretically, polyadenylation extends the miRNA with a poly(A) tail, allowing more dye molecules to intercalate and thereby amplifying the fluorescence signal. In this work, we measured fluorescence spectra of SYBR in aqueous solution, SYBR bound to miRNA (i.e., miRNA-146a, 22 bp) and SYBR bound to extended miRNA obtained from miRNA-146a through polyadenylation (∼150 bp). The results are shown in Fig. 3. Across all three solutions, the maximum fluorescence emission occurs at the same wavelength of 508 nm. Although SYBR staining of miRNA at 100 fM exhibited slight fluorescence enhancement, staining of the polyadenylated miRNA led to a pronounced signal increase. This enhancement likely arises from polyadenylation-induced elongation of the miRNA, which promotes the formation of higher-order structures favorable for SYBR intercalation. These results clearly indicate that significant signal amplification is achieved for detection of miRNA by poly(A) polymerase-catalysed polyadenylation, adding a poly(A) tail at the 3′-end of miRNA. This analytical strategy allows the development of a sensitive assay for miRNAs.

Fig. 3 Fluorescence emission spectra measured from SYBR Green II, miRNA + SYBR Green II, and extended miRNA + SYBR Green II solutions. [miRNA] = 100 fM, [SYBR] = 1 : 50 000, λ_ex = 450 nm.

3.4. Analytical figures of merit

Analytical sensitivity, repeatability, and dynamic range were evaluated for the proposed under the experimental conditions selected. Calibration curves were prepared with miRNA-164a-5p standard solutions at final concentrations ranging from 1.0 fM to 1.10 pM. Triplicate analyses were carried out for each solution. The standard solutions were treated consecutively with ssDNA-MB composites to isolate miRNA-146a-5p from the sample matrix, with poly(A) polymerase and ATP to polyadenylate miRNA146a-5p, with heating to release extended miRNA-146a-5p into solution via denaturing, and with SYBR Green II for fluorescence detection of miRNA-146a-5p. Fluorescence intensity at λ_ex 450 nm/λ_em 508 nm was used for quantification. As shown in Fig. 4A, fluorescence intensity increased proportionally with miRNA-146a-5p concentration, confirming a direct correlation between signal strength and analyte levels. It is noteworthy that most previously reported enzymatic isothermal amplification-based fluorescence assays for miRNA quantification establish calibration curves by correlating fluorescence signals with the logarithm of miRNA concentration. This logarithmic dependence, arising from the mathematical nature of the amplification process, compromises assay accuracy.² These assays employ various isothermal amplification strategies, including rolling circle amplification, duplex-specific nuclease-mediated signal amplification, catalytic hairpin assembly, and the exponential amplification reaction, among others.^19–21,25 The exponential generation of miRNA reporters directly reflects the amplification mechanism. Interestingly, in the assay proposed herein linear regression analysis on fluorescence intensity/analyte concentration data yielded linear calibration curves:

Fluorescence intensity = 0.0059x + 1.79, R² = 0.998

where x is the miRNA-146a-5p concentration in fM. The calibration curve demonstrates a linear relationship between fluorescence intensity and miRNA concentration (Fig. 4B) with a correlation coefficient of 0.998, indicating excellent linearity. The limit of detection (LOD), calculated using the formula LOD = 3.3 × (Sy/slope), is 66.4 fM. The assay is more sensitive than many previously reported fluorescence assays based on duplex-specific nuclease-mediated signal amplification,^30,31 rolling circle amplification,^32,33etc. In addition to fluorescence detection, other detection techniques have also been deployed in PCR-free isothermal quantitative assays for miRNAs such as electrochemical and Raman scattering. Table 2 summarizes several methods reported recently. To assess the assay repeatability, two standard solutions of miRNA-146a-5p (at 250.0 and 750.0 fM, respectively) were analyzed 5 times for each. Repeatability (RSD) of fluorescence intensity was calculated to be 3.6% for miRNA-146a-5p at 250.0 fM, and 5.2% at 750.0 fM, which shows that the proposed method offers good assay repeatability. It should be mentioned that selectivity of the proposed assay is ensured through sequence-selective hybridization between target miRNA and its complimentary ssDNA probe. Our previous study has shown that it exhibits excellent discrimination capability for sequences with single base-mismatch.³⁹ The advantageous analytical performance metrics highlight the present assay's high suitability for sensitive and accurate miRNA quantification in both research and clinical applications.

Fig. 4 (A) Fluorescence emission spectra (at λ_ex 450 nm) obtained from analysing standard miRNA-146a-5p solutions, and (B) calibration curve showing the correlation between miRNA concentration and measured fluorescence intensity (at λ_em 508 nm).

Table 2 Comparing PCR-free miRNA assays based on different techniques

Assay technique	Assay description	Assay sensitivity (LOD)	Samples analyzed	References
Electrochemical nanobiosensor	Decline of square wave voltammetry peak current of the redox probes after hybridization with miRNA	0.28 fM for miRNA-210	Human serum	18
Electrochemical microfluidic biosensor	On-chip miRNA sandwich formation	1.28 nM for miRNA-197	Cultured human serum	34
Electrochemical biosensor	Isothermal amplification based on coupling target-catalyzed hairpin assembly (CHA) with supersandwich formation	0.6 pM for miRNA-221	Serum	35
Surface-enhanced Raman scattering biosensor	Gold nanobipyramid hotspot aggregation-induced SERS in the aid of Exo-III assisted target cycle amplification and TDNs-induced catalytic hairpin assembly amplification	0.59 fM for miRNA-221	Cell lysates	36
Surface-enhanced Raman scattering biosensor	SERS signal on AgNPs in the aid of catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification	42.3 fM for miRNA-21	Blood	37
Surface-enhanced Raman scattering-lateral flow assay	Gold nanocage-modified hairpin DNA sequence and target miRNA-triggered catalytic hairpin assembly (CHA)	2.18 pM for miR-196a-5p	Human urine	38
Fluorescence assay	Fluorescence signal from labels enhanced by Cas12a-based exponential rolling-circle amplification	1.64 fM miR-21	Plasma	19
Fluorescence assay	Fluorescence signal enhanced by nicking-mediated rolling circle amplification coupled with symmetric isothermal circular strand-displacement amplification	5 pM for let-7a miRNA	HeLa cell extracts	20
Fluorescence assay	Fluorescence signal from labels enhanced by the duplex-specific nuclease (DSN)-assisted signal amplification	50 pM for miRNA-21	Cell lysates	31
Fluorescence assay	Fluorescence signal from labels enhanced by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification	1 pM for miRNA-21	Human serum	34
Fluorescence assay	Fluorescence signal from the intercalating dye enhanced by poly(A) polymerase-catalyzed polyadenylation of target miRNA	66.4 fM for miRNA-146a	Cell culture medium and cell lysates	This work

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3.5. Quantification of miRNAs in cellular samples

MDA-MB-231 cells were maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin under standard culture conditions. Culture medium was replaced every 48 hours to ensure optimal cell viability and proliferation. Cell viability was assessed using the Trypan blue exclusion method, and only cultures exhibiting ≥90% viability were used in the tests. Approximately 5 × 10⁶ cells were harvested by enzymatic detachment, collected by centrifugation at 300×g for 5 minutes, and the resulting pellet was transferred to sterile microcentrifuge tubes.

Cell lysis was initiated by adding 100 µL of reaction buffer to each 1 × 10⁶ cell pellet, followed by ultrasonication to achieve efficient disruption of cellular membranes and release of intracellular components. Cell debris was removed by low-speed centrifugation, and the supernatant was carefully transferred to fresh microcentrifuge tubes for miRNA detection. Two cell lines, i.e., HEK-239 kidney normal cells and MDA-MB-231 breast cancer cells were selected for the study. Both intracellular and extracellular (i.e., in culture medium) levels were determined for miRNA-146a-5p and miRNA-10a-5p in these cell lines. The results are shown in Fig. 5A. In HEK-293 kidney normal cells, miRNA-164a-5p is expressed at a significantly lower level than in MDA-MB-231 breast cancer cells, suggesting that malignant transformation influences cellular miRNA expression profiles. It's also noteworthy that in the MDA-MB-231 cellular model, the intracellular level of miRNA-146a-5p is higher than the extracellular level, indicating enhanced intracellular retention or increased miRNA production. This is not the case for HEK-293 normal cells. Target-specific analysis showed differential miRNA expression levels within MDA-MB-231 cells (as shown in Fig. 5B). The intracellular level of miRNA-146a is significantly higher than that of miR-10a-5p. This expression pattern is consistent with the established role of miR-146a-5p in breast cancer pathogenesis, where constitutive NF-κB activation leads to transcriptional upregulation of miR-146a-5p.^40–43 Conversely, miR-10a-5p functions as a tumor suppressor in breast cancer, with significantly lower expression levels observed in the highly aggressive MDA-MB-231 cell line.^44,45 These findings demonstrate that the proposed fluorescence assay can effectively discriminate between miRNAs with different expression profiles in cancer cells, providing quantitative assessment of both high-abundance (miR-146a-5p) and low-abundance (miR-10a-5p) targets.

Fig. 5 Quantification of miRNAs in cellular samples: (A) miRNA-146a-5p in HEK-293 and MDA-MB-231 cells, and (B) intracellular levels of miRNA-146a and miRNA-10a in MDA-MB-231 cells.

By using the proposed fluorescence assay, drug-induced alterations in miRNA-146a-5p expression in MDA-MB-231 breast cancer cells were studied. Paclitaxel is a chemotherapy drug used to treat various types of cancer, including breast cancer. It was selected as the model drug compound in this work. MDA-MB-231 cells were treated with 1 nM and 10 nM paclitaxel for 48 hours. Quantitative analysis of miRNA-146a-5p demonstrated a dose-dependent reduction in its expression in treated cells (Fig. 6). To our knowledge, this represents the first experimental evidence of drug-induced downregulation of miRNA-146a-5p in breast cancer cells.

Fig. 6 Paclitaxel-induced downregulated expression of miRNA-146a-5p in MDA-MB-231 cells.

4. Conclusion

In summary, this work presents a novel label-free fluorescence assay for quantitative detection of microRNAs, based on poly(A) polymerase-catalyzed polyadenylation and SYBR Green II staining. Enzymatic elongation of target miRNAs enables extensive intercalation of SYBR Green II molecules, leading to marked fluorescence enhancement. Unlike most previously reported isothermal amplification-based assays, this label-free system provides a linear calibration relating fluorescence signals with miRNA concentration (R² = 0.998), a low detection limit (66.4 fM), and excellent repeatability. Solid-surface polyadenylation on magnetic beads markedly increased catalytic efficiency and thus, poly(A) tail length compared with conventional solution-phase reactions, thereby improving both assay specificity and fluorescence signal amplification. The assay successfully quantified miRNA-146a-5p and miRNA-10a-5p in both normal (HEK-293) and cancerous (MDA-MB-231) cell lines, revealing expression trends consistent with their known biological functions. Notably, a dose-dependent downregulation of miRNA-146a-5p was observed in paclitaxel-treated MDA-MB-231 cells, representing the first experimental evidence of drug-induced suppression of this oncogenic miRNA in breast cancer cells. Overall, this assay provides a simple, cost-effective, and sensitive method for translational miRNA research and potential clinical biomarker analysis.

Author contributions

Avinash Kumar: data curation, methodology, and writing. Jing Qu: data curation. Yi-Ming Liu: conceptualization, methodology, and writing-reviewing. Yikao Hu: data curation. Xun Liao: conceptualization, methodology, and reviewing.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

The datasets supporting the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

Financial support from US National Institutes of Health (GM145223 to YML) and Natural Science Foundation of Sichuan (2025ZNSFSC1782 to YH) is gratefully acknowledged.

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