Research ArticleBIOCHEMISTRY

Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaax0801
DOI: 10.1126/sciadv.aax0801
  • Fig. 1 Tethering siRNA to the hydrogel via Michael-addition chemistry and its release.

    Schematic illustration of (A) siRNA conjugation to DEX-MAES and (B) hydrogel formation via photocrosslinking and siRNA release upon hydrolytic degradation. (C) Acrylamide gel showing conjugation of siRNA-SH to DEX-MAES over time. (D) Release profiles of unbound (physically trapped siGFP, “unbound siGFP”) and covalently tethered siGFP-SH (“bound siGFP-SH”) from 10% (w/w) DEX hydrogels (20 μg of RNA per 50 μl of gel) (*P < 0.001 compared with “unbound siGFP” at the same time point). UV, ultraviolet; PBS, phosphate-buffered saline; Ds siRNA, double-stranded siRNA; bp, base pairs.

  • Fig. 2 Tethering siRNA to the hydrogel via UV conjugation.

    Schematic illustration of (A) siRNA-MA synthesis and (B) conjugation of siRNA-MA to DEX-MAES via photopolymerization and siRNA release upon hydrolytic degradation of ester and/or disulfide bonds within the DEX hydrogel. (C) Polyacrylamide gels confirming conjugation of siRNA-MA to DEX-MAES after application of UV light. SPDP, succinimidyl 3-(2-pyridyldithio) propionate; DMSO, dimethyl sulfoxide; RT, room temperature.

  • Fig. 3 Release of phototethered siRNA from the hydrogels and RNA bioactivity.

    (A) Release profiles of siRNA (10 μg per 50 μl of gel) from 10% (w/w) DEX hydrogels into phenol red–free DMEM-HG (*P < 0.001 compared with unbound siGFP at the same time point). (B) GFP expression of HeLa cells treated with the same volume of releasates from different groups without the addition of transfection reagent and in the absence of FBS for 2 days (*P < 0.05; #P < 0.05 and &P < 0.05 compared to different time points of the same hydrogels). (C) Concentration of siRNA in releasate at different time points, which were used as transfection media to obtain (B) (*P < 0.05 compared with “bound siGFP-MA” at the same time point). (D) Bioactivity of released siGFP at the same siRNA concentration (350 nM) performed in DMEM-HG containing 0% FBS (“FBS-free”) or 2.5% (v/v) FBS [*P < 0.001 compared to the corresponding (color) FBS groups].

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax0801/DC1

    Table S1. Sequence of the synthesized oligonucleotides.

    Fig. S1. Annealing and gene silencing ability of synthesized siRNA.

    Fig. S2. Chemical structure of DEX-MAES.

    Fig. S3. Schematic illustration of siRNA conjugation to hydrogels via Michael-addition reaction and its release.

    Fig. S4. Effect of hydrogel concentration on the tethered siRNA release behavior and bioactivity of released siRNA.

    Fig. S5. Lipofectamine-mediated bioactivity of released siRNA from siRNA-tethered hydrogels.

    Fig. S6. Transfection reagent-mediated bioactivity of siRNA loaded or tethered in the hydrogels.

    Fig. S7. Schematic illustration of siRNA conjugation to hydrogels via photoconjugation and its release.

    Fig. S8. Additional release profiles of tethered siGFP from hydrogels with longer sampling intervals compared to Fig. 3A.

    Fig. S9. Additional bioactivity data of released siGFP (200 and 350 nM RNA treatment).

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Sequence of the synthesized oligonucleotides.
    • Fig. S1. Annealing and gene silencing ability of synthesized siRNA.
    • Fig. S2. Chemical structure of DEX-MAES.
    • Fig. S3. Schematic illustration of siRNA conjugation to hydrogels via Michael-addition reaction and its release.
    • Fig. S4. Effect of hydrogel concentration on the tethered siRNA release behavior and bioactivity of released siRNA.
    • Fig. S5. Lipofectamine-mediated bioactivity of released siRNA from siRNA-tethered hydrogels.
    • Fig. S6. Transfection reagent-mediated bioactivity of siRNA loaded or tethered in the hydrogels.
    • Fig. S7. Schematic illustration of siRNA conjugation to hydrogels via photoconjugation and its release.
    • Fig. S8. Additional release profiles of tethered siGFP from hydrogels with longer sampling intervals compared to Fig. 3A.
    • Fig. S9. Additional bioactivity data of released siGFP (200 and 350 nM RNA treatment).

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