Self-assembly of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging

We developed a new and highly sensitive method for miRNA detection and intracellular imaging based on novel nucleic acid molecular aggregates self-assembled on graphene oxide nanoplates.

BHQ-DNA6 could hybridize with FAM-DNA5 to partially quench the fluorescence signal. In the presence of GONPs, the fluorescence intensity was dramatically reduced a little of interference might appear in the reaction solution for rolling circle amplification (RSRCA), which would lead to blank value being mildly increased . It might be a reason that a very little of ssDNA-FAM scattered from GONPs.
So, FAM-DNA5 and BHQ-DNA6 hybrids (DHS) were used for reducing blank value, because fluorescence intensity by a very little of DHS scattered from GONPs was very weak (Fig. S2-e). Meanwhile, only when the RCA reaction occurred in the system, BHQ-DNA6 could be replaced by RCA-product, FAM-DNA5 and RCA-product hybrids could generate and fluoresce, which could avoid the interference.

S3. The Triple-helix Probes Immobilized on Carboxyl-modified Magnetic Fluids
The THP molecules immobilized on the surface of CMFs were achieved by some modifications according to the literature that DNA chains were fastened on carboxyl-modified magnetic beads. [S4, S5]

S4. Optimization of the experimental conditions and gel electrophoresis analysis of the RCA products
The amount of Phi29 DNA polymerase was an important influencing factor for the fluorescence intensity. To improve the sensitivity of fluorescence detection, a series of control experiments were designed to optimize the amount of Phi29 DNA polymerase.
As shown in Fig. S5-A, the fluorescence intensities enhanced speedily when the amount of polymerase increased from 0.05 to 0.2 UμL -1 . But after 0.2 UμL -1 , the fluorescence intensity changed mildly. Thus, 0.2 UμL -1 of Phi29 DNA polymerase was chosen to be the optimum. For obtaining the best sensing performance, the incubation time was investigated by implementing the tests of different reaction time.
As illustrated in Fig. S5

S5. Specificity of the assay
It is a great challenge to put into effect the miRNAs assay with high specificity owing to the high sequence homology among family members and small size of miRNAs. So the selectivity of the THP-NAMAs strategy was investigated by four different miRNAs (miR-21, let-7a, let-7b, and let-7c) with the same concentration.
Fluorescence intensity produced by miR-21 is 12.9-, 21.0-, and 12.3-fold of that respectively produced by let-7a, let-7b, and let-7c (Fig. S6). These results suggest that this detection method for miRNAs has high sequence specificity to distinguish the perfectly complementary target and the mismatched sequences, and has promising application in single-nucleotide polymorphism analysis.

Fig. S6
Comparison of the fluorescence intensity produced by 5.0 × 10 -10 M let-7a, let-7b, let-7c, and miR-21, where F and F 0 are fluorescence intensities of amplification products by THP-NAMAs method in the presence and absence of four different miRNAs, respectively. Error bars were estimated from three replicate measurements.

S6. Analysis of the cellular reaction product
The cellular reaction product was detected by the fluorescence intensity in the revised manuscript. Firstly, MCF-7 cells were incubated with culture medium containing DHS on GONPs (25 μg/mL) for 12 h, then were washed three times with PBS (0.01 M, pH 7.4). Secondly, the THP probe and circular-DNA fastened onto CMFs were incubated into the cells. The slides with fixed cells were incubated with 2% formamide for 30 min. After being washed three with PBS, the slides were dehydrated by a series of 70%, 80%, and 99% ice-cold ethanol for 3 min each and air-dried. Thirdly, the RCA reaction was performed in the humidified 37 °C incubator for 90 min with the reaction solution containing DEPC-treated water, 2 μL phi29 DNA polymerase and 6 μL dNTPs. Subsequently, 0.1 mol/L SDS solution was added to the mixture to attain an SDS concentration of 20 mmol/L. [S6] After these processed cells on the slides were lyzed, the mixture was centrifuged (2000 r/min, 6 min) three times, and each supernatant was gathered. Fluorescence measurements of the reaction product were carried out on a F4600 fluorometer (Fig. S7)  The average of three spectra was acquired from different detection, and three repetitive experiments were carried out. Error bars displayed the standard deviation of three experiments. The blank was deducted from each value. Table S1. miRNAs Sequences Used in This Work.

Note
Sequences (