DNA micelle flares for intracellular mRNA imaging and gene therapy

Abstract

Multifunctional DNA micelles: Molecular beacon micelle flares (MBMFs), based on diacyllipid-molecular beacon conjugate (L-MB) self-assembly, have been developed for combined mRNA detection and gene therapy. The advantages of these micelle flares include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high signal-to-background ratio, excellent target selectivity, and superior biocompatibility. In addition, these probes possess a hydrophobic cavity that can be used for additional hydrophobic agents, holding great promise for constructing an all-in-one nucleic acid probe.

Figure 1

Characterization of MBMFs. (a) Structure of MBMFs. Note: Not all the bases are shown. (b) Agarose gel electrophoresis of DNA marker (lane 1), MBs (lane 2), MBs with synthetic complementary target (cDNA) (lane 3), MBMFs (lane 4), and MBMFs with cDNA (lane 5). (c) DLS and (e) zeta-potential measurements of MBMFs, MBMFs with cDNA, and MBMFs with synthetic noncomplementary target (rDNA).

Figure 2

Performance evaluation of MBMFs in buffer system. (a) Fluorescence emission spectroscopy of MBMFs treated with cDNA and rDNA. (b) Fluorescence kinetics spectroscopy of MBMFs treated with cDNA, synthetic 1-base mismatch target (1m-DNA), and synthetic 2-base mismatch target (2m-DNA). (c) Response of MBMFs to cDNA with concentrations ranging from 0 to 1 µM. Inset: Response of MBMFs to cDNA with concentrations ranging from 0 to 200 nM with an excellent linear relationship. (d) Fluorescence kinetics spectroscopy of MBMFs and MBs treated with DNase I.

Figure 3

Capability investigation of MBMFs in living cells. Confocal laser scanning microscopy images of A549 cells treated with 300 nM (a) MBMFs, (b) non-complementary MBMFs, and (c) MBs. Left panels are TAMRA fluorescence pseudo-colored red, and right panels are the overlay of TAMRA fluorescence and bright field image. Scale bars: 20 µm. (d) Flow cytometry results of A549 and HBE135 cells treated with 300 nM MBMFs and non-complementary MBMFs.

Figure 4

Cytotoxicity assay of A549 cells treated with S-MBMFs and non-complementary S-MBMFs.

Scheme 1

Schematic illustration of molecular beacon micelle flares (MBMFs) for intracellular mRNA detection and gene therapy. Diacyllipid-molecular beacon conjugates (L-MBs) self-assemble into MBMFs and enter living cells. Before meeting their target mRNA, the fluorophore and the quencher in MBMFs are in close proximity (“OFF state”). However, hybridization between the loop region and target mRNA separates the fluorophore and the quencher, producing a fluorescence signal (“ON state”) and heteroduplex for RNase H action. Note: In order to maintain the aesthetics of the scheme, not all MBs are shown on the MBMF.