Rapid Detection of MicroRNA by a Silver Nanocluster DNA Probe

LETTER pubs.acs.org/ac

Rapid Detection of MicroRNA by a Silver Nanocluster DNA Probe Seong Wook Yang*,† and Tom Vosch*,‡ †

Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Copenhagen, Denmark ‡ Nano-Science Center/Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark

bS Supporting Information ABSTRACT: MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis. Thus, innovative new tools for rapid, specific, and sensitive detection of miRNAs are an important field of research. Using the fluorescence properties of DNA-nanosilver clusters (DNA/AgNC), we have designed a DNA/AgNC probe that can detect the presence of target miRNA. Here, we show that the red fluorescence of the DNA/AgNC probe is diminished upon the presence of target miRNA without pre- or postmodification, addition of extra enhancer molecules, and labeling. The DNA/AgNC probe emission was lowest when the complementary miRNA target was present and was significantly higher for four other control miRNA sequences. Also, when adding whole plant endogenous RNA to the DNA/AgNC probe, the emission was significantly higher for the mutant where miRNA was deficient. On the basis of these findings, we suggest that these DNA/AgNC probes could be developed into a new, simple, inexpensive, and instant technique for miRNAs detection.

R

ecently, the creation of small silver clusters as new, bright, and photostable labels has received significant attention in literature.1,2 When significantly small (less than 100 atoms) to exclude continuous density of states, discrete transitions between energy levels are possible, allowing for emission to occur. In order to achieve the creation of these small silver clusters and to avoid aggregation into larger nonemissive particles, a myriad of different scaffolds have been used. These scaffolds include biological compounds (e.g., DNA, proteins),3
21 polymers,22
25 dendrimers,26 organic compounds,27
30 and inorganic matrixes.31
35 Here, we have chosen a DNA sequence which has a specific complementary sequence against a target miRNA and a 12 nucleotide scaffold for producing red emitting silver nanoclusters (AgNC). When target miRNA is present, the emission of the DNA-nanosilver clusters (DNA/AgNC) probe is significantly lower versus the case when no target miRNA or other miRNA is present. In addition to conventional methods (e.g., Northern blot analysis) and newly emerging nanotechnology-based methods (such as the use of electrocatalytic nanoparticle tags, surface plasmon resonance imaging, gold-nanoparticles-based array, and surface enhanced raman scattering (SERS)-based assays, illumina deep sequencing),36,37 our DNA/ AgNC probe offers interesting possibilities to become a new, fast, fluorescence-based method for miRNA detection in miRNA research and disease diagnosis.

’ RESULTS AND DISCUSSION The number of reports using DNA sequences to create AgNC has been rapidly increasing in recent years.4
15,17,19
21,38
40 We r 2011 American Chemical Society

therefore looked in the literature for specific DNA sequences that successfully produced red emitters as a scaffold. On the basis of the work of Richards et al., we chose a sequence that successfully created a red emitting AgNC (DNA-12nt-RED: 50 -CCTCCTTCCTCC-30 ).15 DNA-12nt-RED created red emissive AgNCs, with an emission maximum at 620 nm, molar extinction coefficient of 120 000 M
1cm
1, fluorescence decay time of 2.23 ns, and a fluorescence quantum yield of 32%.15 DNA-12nt-RED also outperformed Cy3 by at least 1 order of magnitude in photo stability when monitoring individual molecules embedded in a polymer film.15 Therefore, we believed that the DNA-12nt-RED would be an ideal candidate to be used for developing a DNAbased probe for miRNA detection. As a proof of principle, we designed a DNA/AgNC probe (DNA-12nt-RED-160 probe) for the detection of RNA-miR160 targets. We have chosen miR160 since it targets the transcription of Auxin Response Factor genes which are important for Auxin (plant hormone) signaling in Arabidopsis.41 In order to create the probe for detecting miRNA, the complementary DNA sequence of RNA-miR160 was attached to the DNA-12nt-RED sequence. Figure 1A shows the sequences of DNA-12nt-RED, DNA-160, and the DNA-12nt-RED-160 probe that were used. Just like for DNA-12nt-RED, after addition of AgNO3 and reduction with NaBH4 (DNA/AgNO3/NaBH4 in a 1:17:17 ratio), the DNA-12nt-RED-160 probe displayed strong red emission from the AgNCs. Figure 1B shows the time evolution Received: July 25, 2011 Accepted: August 23, 2011 Published: August 23, 2011 6935

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Figure 1. (A) Sequences of DNA-160, DNA-12nt-RED, and the DNA12nt-RED-160 probe. (B) Excitation and emission spectra of the DNA12nt-RED-160 probe. The emission spectra (excited at 560 nm) were recorded 15, 30, and 60 min after mixing and reducing the DNA-12ntRED-160 probe with AgNO3 and NaBH4. The excitation spectrum (monitored at 620 nm) was recorded 60 min after mixing and reducing the DNA-12nt-RED-160 probe with AgNO3 and NaBH4.

of the fluorescence intensity of the DNA-12nt-RED-160 probe, when excited at 560 nm. Also, an excitation spectrum is given, monitoring the emission at 620 nm. A full spectral scan of the emission as a function of excitation wavelength for the DNA12nt-RED-160 probe can be found in Supporting Information Figure 1. The formation of the red emitting AgNC with the DNA-12nt-RED-160 probe is much faster than for the DNA12nt-RED (1:6:6 ratio)15 sequence, and the maximum emission intensity is more than 100 times brighter after 1 h. For the DNA12nt-RED-160 probe, the fluorescence intensity levels out, one hour after addition of AgNO3 and reduction by NaBH4 (see Supporting Information Figure 2 for a comparison of the fluorescence intensity after 1, 2, and 3 h). For the DNA-12nt-RED sequence, the fluorescence intensity is low after 1 h (see Supporting Information Figure 3A and Supporting Information Table 1), and it takes several hours for the red fluorescence to appear (see Supporting Information Table 1 for a comparison on the AgNC creation speed differences between DNA-12nt-RED and the DNA-12nt-RED-160 probe). The reason why the speed of AgNC formation is faster and the overall fluorescence intensity is about 100 times higher in the DNA-12nt-RED-160 probe versus DNA-12nt-RED is not fully understood. A possible explanation could be a recent finding where Yeh et al. showed that AgNC fluorescence increased 500-fold upon hybridization.4 This would mean in our case that the DNA-12nt-RED-160 probe could form a self-dimer in absence of miRNA. Another alternative explanation could that the DNA-12nt-RED-160 probe forms a hairpin structure, yielding bright emitters.11,42 Currently, we are working on elucidating the reason why this dramatic higher fluorescence intensity is observed in the DNA-12nt-RED160 probe. The DNA-160 sequence by itself was also tested for red AgNC formation. The sequence is also able to form red emitting AgNC (1:17:17 ratio), however less than the DNA12nt-RED sequence (see Supporting Information Figure 3B and Supporting Information Table 1).

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Figure 2. (A) Fluorescence intensity of the AgNCs formed after addition of AgNO3 and NaBH4 to a mixture containing 1.5 μM DNA12nt-RED-160 probe and RNA-miR160 target in a concentration ranging from 0 to 1.5 μM. The fluorescence spectra were recorded, exciting at 560 nm. (B) Stern
Volmer plot of the data presented in panel A.

Figure 3. (A) Sequences of the DNA-12nt-RED-160 probe and 4 miRNA targets: RNA-miR160, RNA-miR166, RNA-miR172, and RNA-RY-1. (B) Emission spectra (excited at 560 nm) of 1.5 μM DNA12nt-RED-160 probe (black curve) and mixtures of 1.5 μM DNA-12ntRED-160 probe with 0.5 μM of RNA-miR160 target (red curve), RNA-miR163 target (blue curve), RNA-miR166 target (green curve), RNA-miR172 target (pink curve), and RNA-RY-1 target (brown curve). (C) I0/I values of the fluorescence intensity of the AgNC when adding 0.5 μM of the target miRNA sequences to 1.5 μM of DNA-12nt-RED160 probe. The values are the average of 3 measurements each.

On the basis of the above-mentioned results, we can conclude that the DNA-12nt-RED-160 probe forms within one hour a large amount of red emitting AgNCs. In a next step, we tested whether we can use the DNA-12nt-RED-160 probe to detect the complementary RNA-miR160 target sequences. Figure 2 demonstrates that the DNA-12nt-RED-160 probe can be used for detecting the RNA-miR160 target molecules by monitoring the generated red fluorescence of the AgNCs. It can be clearly seen in 6936

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Figure 4. (A) Emission spectra (excited at 560 nm) of AgNC emission from a solution containing 1.5 μM of DNA-12nt-RED-160 probe and 20 μg of endogenous RNA from WT (black curve) or hyl1-2 mutant Arabidopsis thaliana plants. (B) The inset shows the I0/I ratio. (C) Northern blot analysis showing the presence and absence of RNAmiR160 in WT and hyl1-2 mutant plants, respectively. The level of U6snRNA was used as a loading control.

Figure 2A that the presence of an increasing concentration of RNA-miR160 sequences leads to a decrease in the observed red fluorescence. The way the experiments in this publication, that involve mixing the DNA-12nt-RED-160 probe with miRNA (including the whole plant RNA), were performed is by first mixing the DNA-12nt-RED-160 probe and miRNA target together, followed by addition of AgNO3 and reduction by NaBH4 and monitoring the red emission after 1 h. An alternative way where first the red emitting AgNCs are created by mixing the DNA-12nt-RED-160 probe with AgNO3 and NaBH4, followed by addition of the miRNA, also works but led to a less significant drop in the fluorescence intensity (see Supporting Information Figure 4 for an example). Hybridization of the RNA-miR160 target with the DNA-12nt-RED-160 probe is the likely cause of the drop in the observed red cluster formation/fluorescence. The exact mechanistic details behind the drop in the observed red cluster formation/fluorescence are currently being studied. Figure 2B shows that the Stern
Volmer plot of the data in Figure 2A follows a linear dependence of the I0/I intensity versus RNA-miR160 target concentration (I0 being the value without addition of RNA-miR160) with a value for the slope of 13. This results in a KD
1 RNA-miR160 target concentration, at which the fluorescence is at 50%, of 76 nM (which corresponds to an amount of 38 picomole in the 500 μL volume). The latter shows that this opens perspectives for picomole detection of miRNA. In a next step, the effect of adding different miRNA target sequences to the DNA-12nt-RED-160 probe was investigated.

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Figure 3 shows an overview of the observed red AgNC fluorescence, 1 h after AgNO3 and NaBH4 are added to solutions containing final concentrations of 1.5 μM DNA-12nt-RED-160 probe and 0.5 μM of RNA-miR160, RNA-miR163, RNAmiR166, RNA-miR172, and RNA-RY-1 target sequences (see Figure 3A for the respective sequences). The RNA-miR160 target has the largest effect on I0/I ratio as can be seen in Figure 3C (a 6 times drop in the fluorescence intensity), while the presence of RNA-miR163, RNA-miR172, or RNA-RY-1 target only has a limited effect on the observed fluorescence intensity of the DNA-12nt-RED-160 probe. The presence of miR166 target leads to a moderate drop in the fluorescence intensity by a factor of 2.2. This moderate drop in the I0/I ratio for the DNA-12nt-RED-160 probe/RNA-miR166 pair could be due to the higher degree of random binding between the DNA12nt-RED-160 probe and the RNA-miR166 target, versus the other tested target sequences. The highest drop in the I0/I ratio was, however, observed for the cDNA-12nt-RED-160 probe/ RNA-miR160 target pair. This clearly opens perspectives toward designing and creating probes with a high specificity toward detecting specific miRNA sequences. As a proof of principle experiment, the DNA-12nt-RED-160 probe was used for detecting the presence of miRNA in whole plant (Arabidopsis thaliana) endogenous RNA. For this detection assay, we used a wild type (WT) plant where microRNAs (miRNAs), including the RNA-miR160 target, are present and a mutant (hyl1-2) which has a defect in the miRNA processing pathway.43 As shown in the Northern blot analysis (Figure 4C), the level of RNA-miR160 in the hyl1-2 plants is dramatically reduced compared to the level in WT plants. As a loading control, the presence of U6 small nuclear RNA (U6 snRNA), which is not related to the miRNA processing pathway, was checked by Northern blot analysis and the level was similar for both WT and hyl1-2 (Figure 4C). We observed a notable reduction of fluorescence when the DNA-12nt-RED-160 probe was mixed with the endogenous RNA from the WT plants. As shown in Figure 4B, the fluorescence dropped a factor of 30 compared to the DNA-12nt-RED-160 probe without any plant RNA addition. The observed intensity levels are similar to these of the control experiment where endogenous plant RNA was mixed with AgNO3 and NaBH4 without the presence of the DNA-12ntRED-160 probe (see Supporting Information Figure 5 for the emission spectra of the WT and hyl1-2 case). The addition of endogenous RNA from hyl1-2 showed a 4 times higher intensity of fluorescence than for the WT case. The result of our detection assay is consistent with the results of the Northern blot analysis (Figure 4C), clearly showing the possibility to detect the presence of miRNA. We must however also conclude that a part of the drop in the red fluorescence of the DNA-12nt-RED-160 probe is not due to the specific interaction with the RNA-miR160 target but is probably due to interaction with nonspecific small RNAs, intermediate RNAs, and other miRNAs or unknown biological materials. The latter can be assumed from the observed I0/I value of 7.5 when adding the endogenous RNAs of hyl1-2 plants to the DNA-12nt-RED-160 probe where hardly any traceable amount of miRNA is present. Also, we do not expect that the amount of RNA-miR160 present in the endogenous WT plant RNA (20 μg, which is a normal amount used for small RNA Northern blot analysis) is sufficient to increase the I0/I value to 30, which would require the presence of a 2 μM concentration of RNA-miR160, according to the Stern
Volmer plot (Figure 2). 6937

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Analytical Chemistry Nevertheless, we have shown here two important facts. First, the fluorescence of the DNA-12nt-RED-miR160 probe forms a very strong fluorescence within an hour and is still sufficiently high in the presence of all the background of endogenous RNAs from hyl1-2 plants, leaving enough room for the detection of miRNAs by a drop in the fluorescence intensity. Second, our method was based on the same logic of small RNA detection by Northern blot analysis and it displayed a consistent similar tendency with it. Even though our fluorescence-based detection assays were performed at 25 °C without washing procedures (small RNA Northern blot analysis has the best stringency for target miRNAs at 42 °C with a washing procedure), we can conclude that this simple fluorescence-based miRNA detection scheme in the presence of endogenous RNA is a remarkable achievement and we speculate that the addition of washing steps at 42 °C to our procedure could further enhance target specificity (e.g., RNAmiR160) for our method.

’ CONCLUSIONS In this Letter, we have demonstrated the proof of principle that the drop in AgNC emission from DNA stabilized AgNCs can be used as a probe for detecting the presence of complementary miRNA sequences. Moreover, we have shown that the DNA12nt-RED-160 probe for detecting RNA-miR160 rapidly formed a strong fluorescence from red emitting AgNCs within an hour. Also, the fluorescence of the DNA-12nt-RED-160 probe was high enough to be able to detect a clear signal in the presence of a miRNA free, nonspecific background of total plant RNA. We have shown here only the method for plant RNA-miR160 detection, but further development of our suggestions for miRNA detection may be useful for biological research and diagnostic applications in both plants and animals. For instance, the expression levels of miRNAs are correlated to metastatic potentials, therapeutic responses, and clinical status in various types of cancer.44 Although the presented results here indicate that high specificity toward detecting specific miRNA sequences is possible, a more thorough investigation with a larger set of target miRNA sequences and adequate designing of relevant DNA probes and procedure optimization (e.g., hybridization and washing step at 42 °C) must be performed. When high specificity is achieved, this fluorescence-based technique would be a much faster (1 h versus days), fluorimeter-based alternative to Northern blotting and would not require the use of radio-isotopes or DIG labeling for miRNA detection. In addition, the detailed mechanism of the observed fluorescence drop upon DNA probe/target miRNA interaction should be further investigated to optimize the method for miRNA detection. On the basis of the results presented here, we are currently investigating these perspectives for developing this AgNC-based fluorescence method into a new technology for miRNA detection. ’ EXPERIMENTAL SECTION AgNO3 (99.9999%) and NaBH4 (99.99%) were purchased and used as received from Sigma Aldrich. MiRNA targets and DNA probes were commercially synthesized by Eurofin. Fluorescence spectra were recorded by a fluorimeter (Horiba Jobin Yvon, Fluoromax-4) in a 1 mm quartz cuvette. Purification of small RNAs of WT and hyl1-2 plants and Northern blot analysis were performed as described in Yang et al.45 A detailed synthesis protocol for making the fluorescent AgNCs can be found in the Supporting Information.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Synthesis procedures for the fluorescent AgNCs, additional spectra on AgNC emission in the DNA-12nt-RED-160 probe and DNA-12nt-RED and DNA-160 sequence. A table comparing fluorescence intensity and time evolution for the DNA-12nt-RED-160 probe and DNA-12nt-RED and DNA-160 sequence. Emission spectra of endogenous plant (Arabidopsis thaliana) RNA (WT and hyl1-2 mutant) mixed with AgNO3 and NaBH4. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (S.W.Y.) and [email protected] (T.V.).

’ ACKNOWLEDGMENT S.W.Y and T.V. gratefully acknowledge financial support from the “Center for Synthetic Biology” at Copenhagen University funded by the UNIK research initiative of the Danish Ministry of Science, Technology and Innovation (Grant 09-065274). We thank Thomas G€unther-Pomorski for use of his fluorimeter which is supported by Carlsbergfondet and Bj€orn Hamberger for discussions. ’ REFERENCES (1) Díez, I.; Ras, R. H. A. In Advanced Fluorescence Reporters in Chemistry and Biology II; Springer: Berlin Heidelberg, 2011; Vol. 9, pp 307
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