Research ArticleHEALTH AND MEDICINE

A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

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Science Advances  01 Apr 2020:
Vol. 6, no. 14, eaay2801
DOI: 10.1126/sciadv.aay2801
  • Fig. 1 Met-binding DNA aptamers and their nuclease stability.

    (A and B) Schematic representation of HGF-induced Met activation and the Met activation potential of the Apt-mono and the Apt-dimer. (C) (Top) Schematic representation of the oligonucleotides used in the present work. (Bottom) Nuclease stability of the oligonucleotides in serum. Each oligonucleotide (2 μM) was incubated in PBS containing 50% FBS at 37°C. (D) Sequence and predicted secondary structure of the aptamers used in the present work. The sequence of the Apt-mono (purple) was identified as the minimal binding motif of a Met-binding aptamer (CLN0003) reported previously (22). The Apt-dimer [termed “ss-0” in a previous work (11)] was designed as a tandem dimer of the Met-binding aptamer, as depicted in the figure. The stem sequence of the 3′-mono (green) was replaced with an alternative complimentary sequence to prevent the misfolding of the aptamer.

  • Fig. 2 Design of an unfunctional mutant of the Apt-dimer (G4 mutant) and microdistribution of the oligonucleotides in liver tissues.

    (A) Sequences of the oligonucleotides used in the experiments depicted in Fig. 2. The four G-to-A mutations of the G4 mutant are highlighted. (B) Nuclease stability of the Apt-dimer and G4 mutant. Each oligonucleotide (2 μM) was incubated in PBS containing 50% FBS at 37°C. After incubation, the samples were immediately analyzed using denaturing 15% PAGE. (C) Western blotting analysis of the phosphorylation level of Met in SCCVII cells after 15 min of incubation with oligonucleotides. (D) Confocal imaging of liver tissues. Alexa Fluor 647 carboxylic acid (0.5 nmol) or a 5′-Alexa Fluor 647–labeled oligonucleotide (0.5 nmol) was intravenously injected (red). Blood vessels were visualized by injecting 25 μg of DyLight 488–conjugated tomato lectin (green). Ten minutes after the oligonucleotide injection, the liver was excised and observed directly using a confocal laser scanning microscope. Scale bars, 50 μm.

  • Fig. 3 Intravital real-time imaging of liver tissues after systemic injection of the oligonucleotides.

    (A) 5′-Alexa Fluor 647–labeled Apt-dimer (0.5 nmol), (B) 5′-Alexa Fluor 647–labeled G4 mutant (0.5 nmol), or (C) Alexa Fluor 647 (0.5 nmol) was intravenously injected. (Left) Confocal images. Lower panels show enlarged and brighter images of the boxed region in the upper panels. The dotted circles highlight the leakage of Apt-dimer to the liver parenchyma. (Right) Intensity profile of Alexa Fluor 647 in the region indicated by the white arrows in the left figures. Scale bars, 50 μm. a.u., arbitrary units.

  • Fig. 4 Tissue distribution of the aptamer.

    Tissue distribution of the 5′-Alexa Fluor 647–labeled oligonucleotides. (Left) Representative ex vivo fluorescence image of the organs 10 min after intravenous injection of Alexa Fluor 647–labeled oligonucleotides (0.5 nmol). The organs were excised after perfusion with PBS and imaged using IVIS (excitation/emission = 640/680 nm). (Right) Biodistribution of oligonucleotides quantified by fluorescence intensity in the supernatant of the organ homogenates. The results are expressed as the mean ± SD (n = 3). Statistical significance was examined by two-sided Student’s t test (**P < 0.01; *P < 0.05).

  • Fig. 5 Agonistic functions and therapeutic potential of the HGF-mimic aptamer.

    (A) Schematic representation of the experimental outlines. (B) Immunoblotting analysis of Met activation in mouse livers after intravenous injection of recombinant human HGF (rhHGF) or oligonucleotides. The liver was excised 10 min after the intravenous injection of rhHGF (1 μg) or oligonucleotides (1 to 10 nmol). (C) Immunohistochemistry of Met activation in the mouse liver after intravenous injection of the oligonucleotides. The liver was excised 10 min after the intravenous injection of oligonucleotides (10 nmol). (D) (Left) Schematic representation of the apoptotic signal induced by Fas-Ab and the antiapoptotic signal induced by Met activation. (Right) Effect of Apt-dimer administration on a fulminant hepatitis mouse model. An anti–Fas-Ab (2 ng) was co-injected with vehicle control (n = 10), Apt-dimer (10 nmol, n = 9), or G4 mutant (10 nmol, n = 9) intravenously. The oligonucleotide (10 nmol) was administered 1.5 hours later. The average activity of glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), and lactate dehydrogenase (LDH) is shown with error bars (SD). Statistical significance was examined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (***P < 0.001; **P < 0.01; ns, not significant).

Supplementary Materials

  • Supplementary Materials

    A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

    Ryosuke Ueki, Satoshi Uchida, Naoto Kanda, Naoki Yamada, Ayaka Ueki, Momoko Akiyama, Kazuko Toh, Horacio Cabral, Shinsuke Sando

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