Design of a Low-cost Hydrogel made of RNA-origami for 3D Bioprinting Applications
Authors: Raqbi Karim , Tomita Masaru , Galipon Josephine 
Published: February 2020

Hydrogels are three-dimensional polymer networks that swell to some extent in aqueous solutions (Ahmed et al. 2015). Their physical properties allow for applications ranging from contact lenses, wound dressing, drug delivery and tissue engineering (Calo et al. 2015). The latter application requires biocompatible and biodegradable hydrogels that are designed to have sufficient mechanical strength and stability in human physiological conditions to allow time for cells to build their own scaffold.
Nucleic acid-based hydrogels have already proven their efficiency and a variety of designs have been explored since the first deoxyribonucleic acid (DNA) hydrogel was published (Um et al. 2006). DNA-hydrogels were reported to assemble using mixtures of anywhere from 1 to 5 oligomers. For instance, 3 oligomers forming a Y-shape structure are mixed with a linker solution consisting of 2 complementary oligomers with overhangs that may bridge the Y-shapes together (Xing et al. 2011). DNA-origamis may also be generated from stapling the single-stranded genome of the M13 phage with short DNA oligomers to make it to adopt pre-designed 2D- or 3D-structures (Rothemund et al. 2006, Dietz et al. 2009). However, the production of single-stranded DNA remains extremely costly and most of these experiments cannot be conducted on the larger scales necessary to assess biocompatibility with cell cultures.
Ribonucleic acid (RNA), an intermediary compound in the genetic flow of information is mainly produced in single-stranded form, and may be mass-produced in any laboratory by in vitro transcription using recombinant T7 RNA polymerase from bacteriophage, and a double-stranded DNA template obtained by molecular cloning. The use of an RNA-hydrogel would therefore allow for properties similar to those of DNA-hydrogels at a fraction of the cost.
So far, only one study succeeded in producing what they call an RNA hydrogel (Huang et al. 2017). They report the chance discovery of an RNA aptamer that self-assembles into a viscoelastic solution at low temperature (5°C). Ideally, the hydrogel should exist at body temperature (37ºC) and gelation should be controllable by some trigger, such as pH, temperature, light, or the mixing of two solutions for downstream 3D printing applications. We designed a simple X-structure using 2 RNA oligomers that bind to each other using a complementary sequence. This type of configuration was reported previously for a DNA hydrogel called “Takumi” using 33-mers that bind to each other by a 13-nt complementary sequence (Nishida et al. 2016).
Using custom code and biopython (Cock et al. 2009), we generated 2,000 random RNA blocks of 20 nucleotides (nt) with melting temperatures between 58°C and 62°C to increase our chances of obtaining a gel that is stable at physiological temperature. These building blocks of 20nt were assembled at random into 60nt-long oligomers to form a library. Each oligo was associated with a partner for which the middle 20nt block is the reverse complement sequence (Fig. 1A). The sequences were then tested using RNAfold included in the Galaxy RNA Workbench (Access et al. 2011, Afgan et al. 2016) and the sequences sorted by folding energy (∆G) to rule out intramolecular secondary structures. The best candidate RNA oligomer pairs were double-checked using the freely available OligoCalc (Kibbe et al. 2007) and Multiple Primer Dimer Analyzer (Thermo Fisher Scientific, Inc.) to rule out any RNA oligomers with unwanted intra- or intermolecular structures. Finally, we modelled the binding of the two RNA oligomers using simRNA (Bioniecki et al. 2015), a coarse-grained model specialized for RNA structures. The different conformations clustered to show possible interaction behaviors in 3D. Fig. 1C shows the 3D model of the sequences we finally selected.
The design was first tested with DNA oligomers mixed at an equimolar concentration of 500µM each. The mixture was then subjected to a step-down temperature gradient in a thermocycler, starting at 95°C for 5 min and cooled down in steps of 2°C every 3min20s until the solution reached room temperature (21°C). The solution obtained was viscoelastic and remained on one side of the tube after flicking. We are currently working on the optimization of the gelation process and the mass production of RNA.

Figure 1