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J. Name ., 2013, 00, 1-3 | 1
Please do not adjust margins Received 00th January 20xx, Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x /
A Ligation-Based Loop-Mediated Isothermal Amplification
(Ligation-LAMP) Strategy for Highly Selective MicroRNA Detection
Wenfang Du, Mengmei Lv, Junjie Li, Ruqin Yu*, and Jianhui Jiang*
A novel ligation-based loop-mediated isothermal amplification (Ligation-LAMP) method has been developed for miRNA detection, which enables highly selective and sensitive quantitative detection of miR-21 in a dynamic range from 1 fM to 1 nM with the ability of discriminating single-base mismatch.
MicroRNAs (miRNAs) are critical to many biological processes and play crucial roles in post-transcriptional regulation of gene expression.1 Recent studies have found that aberrant expression of miRNAs is closely correlated with various diseases such as cancers,2,3 identifying miRNAs as promising theranostic biomarkers for these diseases.4 Recent years have witnessed increasing interest in development of strategies for miRNA detection.5-7 However, quantitative detection of miRNAs with high robustness, sensitivity and selectivity is still a challenge on account of their inherent characteristics such as short lengths, vulnerability to degradation, similarities of sequences and low ab
undance.8,9 Therefore, development of new strategies for miRNA detection is essential for miRNA based biomedicine. The most widely used methods for miRNA detection are Northern blotting 10 and DNA microarrays 11. These methods typically show insufficient sensitivity and require a large amount of samples. Nucleic acid amplification strategies provide an invaluable tool for miRNA detection because of their advantages in detection of miRNA at very low abundance. These strategies include quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR),12 rolling circle amplification (RCA),13,14 duplex-specific nuclease signal amplification (DSNSA),15,16 enzymatic repairing amplification (ERA),17 strand displacement amplification (SDA),18 and loop-mediated isothermal amplification (LAMP).19 However, these techniques still exhibit inferior selectivity in discriminating miRNA sequences of high similarity, since they do not incorporate a mechanism for high fidelity identification of sequence variations. It is known that subtle mutations in miRNA can reduce or eliminate binding to key targets or even drastically change its specificity, implying the importance of mutation detection for miRNA analysis.20 Hence, the pursuit of highly selective and ultrasensitive approach for miRNA
detection is highly demanding.  Herein, we develop a highly selective miRNA detection strategy based on reverse transcription of target miRNA into cDNA and cDNA templated ligation of two hairpin probes
followed by a LAMP mediated detection of the ligated product. A unique design in this strategy is the use of high fidelity ligase to construct a template that can be efficiently amplified utilizing LAMP reaction. It is known that LAMP is a highly efficient nucleic acid amplification technique performed under isothermal conditions using only a DNA polymerase with strand displacement activity.21,22 However, there is no study on its improved versions that enable highly selective discrimination of single nucleotide mutation. LAMP was also reported for miRNA detection based on miRNA-primed
synthesis of a dumb-bell initiator for LAMP reaction.19 However, this method may have relatively high background due to high reaction temperature facilitated stand displacement with backward primers, and show compromised mutation detection ability because of poor discrimination for some mismatches in primer extension reactions.23 To develop a highly sensitive and specific miRNA detection strategy, we reason that the high fidelity of ligase in discriminating single-base mismatch may be exploited. Motivated by this hypothesis, we design a novel ligation based LAMP (Ligation-LAMP) strategy that for the first time utilizes a high-fidelity DNA ligase, Taq  DNA ligase,24 in the construction of dumb-bell DNA initiator for LAMP reaction, as shown in Scheme 1. The miRNA target is firstly extended with poly(A) polymerase to produce a
poly(A) tail at 3' end followed by reverse transcription with M-MuLV reverse transcriptase using a DNA
primer. The poly(A) tail to miRNA target is introduced here to allow duplicating most of 3’ region of the miRNA sequence in its cDNA target such that mutations in the 3’ region can be identified in our Page 1 of 4
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Please do not adjust margins  Scheme 1 Schematic illustration of the Ligation-LAMP strategy
for highly selective miRNA detection
assay. The DNA primer, designed according to the mutation to be identified, includes two domains, one domain complementary with 3' end of target miRNA, and the other being T nucleotides. Because the DNA primer has a domain specific for target miRNA, only target miRNA can be reversely transcribed by M-MuLV reverse transcriptase, yielding a cDNA of target miRNA. The intrinsic RNase H activity of M-MuLV
reverse transcriptase then mediates the degradation of miRNA target in the cDNA:miRNA duplex, producing a single-stranded cDNA as the template for ligation. Two hairpin DNA probes (H1 and H2) are designed for constructing the dumb-bell DNA initiator. Probe H1 has an overhang complementary to the half
fragment of cDNA at 3’ end, with its 3’ termini overlapping the polymorphic sites in cDNA or miRNA. Probe H2 has an overhang complementary to the other half fragment of cDNA at 5’ end. In the presenc
e of cDNA reversely transcribed from target miRNA, probes H1 and H2 can anneal on the cDNA. A perfect match between the base at 3’ end of probe H1 and cDNA allows covalent ligation of these two probes in the presence of Taq  DNA ligase. Because of the high-fidelity of the ligase, a mismatch will disable ligation of these two probes. Therefore, only in case when the cDNA has a half fragment perfectly matching the overhang of H1, a ligated product of probes H1 and H2 can be obtained, affording high specificity in identifying the mutations in target miRNA. The ligated product has a dumb-bell shape, which can act as the initiator for an efficient LAMP reaction using two primers in the presence of Bst  DNA polymerase.21 Such a LAMP reaction is able to produce a large number of long double-strand DNA replicates, achieving an exponential amplification of the ligated product and delivering an intense fluorescence in the presence of SYBR Green (SG) I,25 a selective double-stranded DNA staining dye. In the absence of cDNA reversely transcribed from target miRNA or there is a mismatch between cDNA and the overhang of H1, the dumb-bell structured ligation product cannot form, precluding the LAMP reaction and thus merely
Fig. 1 (A) Real-time fluorescence curves obtained in ligation-LAMP. (a) 1 nM miR-21; (b) 1 nM miR-141, (c) control for 1 nM
miR-21 without reverse transcriptase; (d) control using 1 nM RNA with complementary sequence of mi
R-21 (cmiR-21) without reverse transcriptase; (e) control without miRNA target; (f) control with no Taq  DNA ligase; (g) control without H1 and H2; (h) control without FP and BP; (i) control without Bst  DNA polymerase. (B) Corresponding gel electrophoresis image for a-i. Lane M: DNA marker (10-300 bp).
giving a low fluorescence. Compared with the reported LAMP strategy for miRNA detection,19 the use of high-fidelity ligase in constructing the dumb-bell LAMP initiator provides better discrimination of mutations in miRNA. Moreover, this highly specific ligation reaction is able to minimize the background in synthesis of dumb-bell initiator. Hence, the developed strategy can provide a highly selective and sensitive platform for miRNA detection.
To verify the feasibility of this approach for miRNA detection, the real-time fluorescence intensity curves were investigated for different systems (Fig. 1A) using miR-21 as the case of study. MiR-21 is a known biomarker overexpressed in many cancers,26
and its concentration is in the range from 0.01 pM to 10 pM in cell lysate samples (104 cells/50 μL).17
We observed that in the presence of target miR-21, the fluorescence signal exhibited a typical sigmoidal curve with an abrupt increase within 10 min and a plateau after 35 min (curve a). In contrast, in the absence of miR-21, the fluorescence intensity curve showed a substantial increase after 25 min,
which was 15 min later than that for the positive sample (curve e). In a control experiment in which miR-21 was replaced by miR-141, the obtained fluorescence intensity curve did not have remarkable variation from that obtained with the blank, which suggested the high selectivity of the developed strategy to miR-21 (curve b). A further control experiment with no addition of M-MuLV reverse transcriptase in synthesis of cDNA also gave a fluorescence intensity curve very close to that for the blank (curve c). This finding indicated that reverse transcription synthesis of cDNA was essential for our assay. Moreover, a control experiment using a synthetic RNA (cmiR-21) with the same sequence as cDNA, we also obtained a fluorescence intensity curve with little difference from that for the blank (d). This observation implied the specificity of Taq  DNA ligase in DNA-templated ligation reaction. Additional controls with no Taq  DNA ligase or probes H1 and H2, we found that the real-time fluorescence intensity curves (f and g) showed slower increase at the moments even
later than the blank. The slightly earlier increase in curve e Page 2 of 4
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compared to curves f and g was ascribed to DNA ligase
induced non-specific ligation of DNA probes. We also observed no fluorescence intensity increases (curves h and i) when primers or DNA polymerase was not added. The results evidenced that the slow fluorescence increases in curves f and g were due to DNA polymerase mediated non-specific extension with the probes. Taken together, these findings revealed that the presence of target miRNA was responsible for the early abrupt increase of the fluorescence intensity, indicating that the POI (point of inflection), the time corresponding to the maximum slope in the real-time fluorescence intensity curve,27,28 could be used as the measure for quantitative analysis of miR-21. According to the previous data, the POI values for 1 nM target miRNA and the blank were 17.5 min and 33.5 min, respectively, affording sufficient discrimination between the positive sample and the blank. In addition to the discrimination of POI values, it was also possible to discriminate the positive sample and the blank using the fluorescence spectra at 30 min (Fig. S1, ESI†). It was observed that the fluorescence peak intensity obtained in the presence of 1 nM miR-21 was 3.8-fold and 3.4-fold, respectively, higher than those obtained with the blank and 1 nM miR-141. Further evidences for the discrimination between the positive sample and the blank could be obtained using agarose gel electrophoresis analysis. We found that products with large molecular weight were only obtained in the Ligation-LAMP reaction with target miR-21 (Fig. 1B). Moreover, the ligation products were only found in the presence of perfect matched target and Taq  DNA ligase (Fig. S2, ESI†). These results confirmed the ability of the Ligation-LAMP strategy for selective detection of miRNA.
Having investigated the feasibility of the ligation-LAMP strategy for miRNA detection, we then aimed at optimizing the reaction conditions for the assay. Because the activity of  Bst  DNA polymerase and the hybridization efficiency of nucleic acids were highly dependent upon the temperature, the reaction temperature might have a great effect on the assay. The effect of temperature was then investigated by comparing the fluorescence curves of 1 pM miR-21 with the blank at different temperatures (Fig. S3, ESI†). It was found that the difference of the POI values for the positive and the blank was maximized at 63 ˚C, which was then chosen for the following experiments. Moreover, the influences of the concentrations of hairpin probes, primers, and dNTPs were also studied. The maximum difference of the POI values for the positive and the blank was obtained with 1 nM hairpin probes (Fig. S4, ESI†), which was selected for further use. Similarly, 0.3 μM primers (Fig. S5, ESI†) and 0.6 mM dNTPs (Fig. S6, ESI†) were selected and used in the subsequent studies.
Under the optimized conditions, miR-21 with different concentrations was measured to examine the ability of the Ligation-LAMP system for quantitative detection of miRNA (Fig. 2A). It was observed that the real-time fluorescence curves were all in sigmoidal shape for various concentrations of miR-21, and the POI values decreased gradually with increasing miR-21 concentrations. The POI values were found to exhibit a linear correlation with the logarithmic concentrations over a
Fig. 2 (A) Real-time fluorescence curves of Ligation-LAMP assay for miR-21 of different concentrations. (B) POI values versus logarithmic miRNA concentrations. Error bars are standard deviations of three repetitive experiments.
range from 1 fM to 1 nM (Fig. 2B). The calibration equation was POI = - 6.1201 - 2.6165 lg C with a correlation coefficient R 2 of 0.9953, where C is the concentration of miRNA (M). The detection limit was estimated to be 0.2 fM (2 zmol) based on three times the standard deviation over the background, which was comparable to those for previously reported fluorescence assays for miRNA.16,29,30 Our design provides 500-fold sensitivity enhancement than the previous LAMP assay.19 This high sensitivity implied that the ligation-LAMP strategy held great potential for miRNA-based early diagnosis of diseases. Moreover, the detection performance of this method was also compared to other assays (Table S2, ESI†).
To investigate the specificity of the Ligation-LAMP method for miRNA detection, miR-141, miR-7a, two-base mismatched miR-21 (2M miR-21) and single-base mismatched miR-21 (SM miR-21) were tested (Fig. 3). The ΔPOI (the difference of the POI values between the positive and the blank) of miR-141 and let-7a were almost the same as that with the blank. The ΔPOI values for SM miR-21 and 2M miR-21 were much smaller than that for miR-21. According to the calibration curve (Fig. 2B), the ΔPOI valu
es for SM miR-21 and 2M miR-21 were calculated to amount to 3 fM and 0.9 fM miR-21. These data implied that the developed method gave discrimination ratios over 300 and 1000, respectively, in detection of single-base and two-base mismatches, which indicated high selectivity of the ligation-LAMP assay for miRNA detection. Furthermore, it was found that the ΔPOI obtained for the mixture of miR-21, miR-141 and let-7a was about the same with only miR-21, further verifying the high selectivity of the developed method. The high selectivity of this method might be attributed to high fidelity of Taq  DNA ligase in discrimination toward single-base mismatch. As a matter of
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Please do not adjust margins  Fig. 3 Selectivity of the Ligation-LAMP assay for miRNA detection. MiRNA concentration is 1 pM. Error bars are standard deviation of three repetitive experiments.
fact, the specificity of the Ligation-LAMP method using T4 DNA
ligase was also tested (Fig. S7, ESI†), which exhibited poorer fidelity in discriminating two-base and single-base mismatches than that using Taq DNA ligase.
The validity of our strategy was further evaluated in total
RNA extracts from four human cancer cell lines, including MCF-10A, HT 1080, HeLa, and MCF-7. The results revealed that the determined relative expression levels of miR-21 in the cell lysates were in good agreement with those obtained using qRT-PCR (Fig. S8, ESI†). These results also showed that miR-21
had different expression levels in different cell lines, which was
consistent with previous reports.31 Therefore, the proposed method might hold a great potential for mi
RNA detection in complex biological samples, affording a useful platform for miRNA based diagnostics.
In summary, we have developed a highly selective miRNA
detection strategy based on reverse transcription of target miRNA into cDNA and cDNA templated ligation of two hairpin probes followed by LAMP mediated detection of the ligated product. Compared with the conventional LAMP reaction,21 a
unique design in this strategy is the use of high fidelity ligase to construct the dumb-bell DNA initiator using two hairpin probes in the presence of target miRNA. The dumb-bell DNA initiator can then be efficiently amplified and detected using fluorescence staining reagent in LAMP reactions. The Ligation-LAMP also provides a useful approach to improved
conventional LAMP reaction for highly selective discrimination of single-base mismatches. This method is demonstrated to allow quantitative detection of miR-21 in a dynamic range from 1 fM to 1 nM with a detection limit of 0.2 fM. Moreover,
this assay has the ability of discriminating single-base
mismatch with a discrimination ratio over 300, and shows good performance for miRNA detection in co
mplex biological samples. Therefore, the proposed strategy may provide an
isothermal and cost-effective approach for highly selective and sensitive detection of miRNA, affording useful platform for
miRNA based diagnostics.
This work was supported by the National Natural Science Foundation of China (21527810, 21190041, 21521063).
Notes and references
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