Protein scaffolded DNA tetrads enable efficient delivery and ultrasensitive imaging of miRNA through crosslinking hybridization chain reaction† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc01001c

DNA tetrads as an integrated platform that enables realization of efficient DNA delivery and HCR amplification in situ on nanostructures.

Electrophoresis was performed at a constant voltage of 100 V for 2 h with a load of 10 µL of sample in each lane. After electrophoresis, the gel was visualized using a Tocan 240 gel imaging system (Tocan Biotechnol. Co., Shanghai, China).

In vitro fluorescence assay
To prepare DNA hairpins for DNA tetrads, the all hairpins were kept at 95 °C for 5 min, then quickly treated with ice for 5 min, and finally transferred to room temperature for 2 h before further use. 1x PBS solution was used to dissolve the hairpins. Subsequently, appropriate SA were added to form DNA tetrads. DNA tetrads was incubated with RNA target of a varied concentration in 50 L 1×TAE-Mg 2+ buffer at 37 °C for 4 h. Fluorescence spectra were recorded using a Fluorescence spectrometer (Jobin Yvon Horiba) with excitation at 540 nm. The slit was set to be 5 nm for both the excitation and the emission.
To evaluate its selectivity, the DNA tetrads was also used for detecting other co-existing cellular components including miR-210, miR-214, let-7a, 100 nM β-actin protein, L-02 cells lysate. Additionally, one more sample was incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Cell culture and confocal microscopy imaging
HeLa cells and L-02 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U mL -1 penicillin, and 100 U mL -1 streptomycin. HepG2 cells were incubated in DMEM supplemented with 10% fetal bovine serum.
All cell lines were maintained at 37 °C in a 100% humidified atmosphere containing 5% CO 2 . The cell density was determined using a TC20 automated cell counter (BIO-RAD, USA).
Fluorescence imaging of cells was performed as follows: The cells were washed three times with 1x PBS, then incubated with a fresh medium containing the given amount of DNA tetrads or Lipofectamine 3000 at 37 °C for 4 h. After washing three times with 1x PBS, the cells were incubated with 1 mL fresh medium at 37 °C before imaging.
Fluorescence imaging of lysosome escape of DNA tetrads was performed as follows: HeLa cells were incubated with 500 L culture medium containing the given amount of DNA tetrads at 37 °C for varying time. After washing three times with 1x PBS, the cells were incubated with fresh RPMI 1640 medium containing 50 nM Lysosome traker (Lyso@tracker Red) for 10 min followed by imaging.
Fluorescence imaging of cellular uptake for DNA tetrads was performed as follows: Cells were incubated with 500 L culture medium containing the given amount of DNA tetrads at 37 °C for 1 h. The cells were washed three times with 1x PBS before imaging. For cellular uptake experiment at 4 °C, cells were first incubated at 4 °C for 0.5 h and then grown in 500 L culture medium containing the given amount of DNA tetrads at 4 °C for 1 h followed by imaging. For cellular uptake experiment in the presence of NaN 3 , cells were first pretreated at 37 °C for 1 h with 1 mL culture medium containing 1% NaN 3 and then incubated with 500 L culture medium containing the given amount of DNA tetrads and 1% NaN 3 at 37 °C for 1 h followed by imaging.
All fluorescence images were acquired using an oil dipping objective on Nikon TI-E+A1 SI confocal laser scanning microscope (Japan). Fluorescence of Cy3 and LysoTracker Red DND-99 was obtained by excitation at 561 nm and collected over 570-620 nm; Fluorescence of Cy5 was obtained by excitation at 640 nm and measured over 670-720 nm; Fluorescence of FRET was obtained by excitation at 561 nm and measured over 670-720 nm.

qRT-PCR quantification of miR-21 expression
Total cellular RNA was extracted from HepG2, HeLa, L-02 cells using the SanPrep Column microRNA Mini-Preps Kit  Hairpin probe H1 Biotin-TTT TTT TTT TTT TTT TTC Figure S1. 3% Agarose gel to compare the purity of the DNA nanotetrads before and after agarose gel purification.