Editor’s note: This discovery brings scientists one step closer to creating significantly cheaper and more stable synthetic nucleic acids for use in medicine, materials science, and other fields of research.
Editor: Dr. Shane Friesen
Nucleic acids, such as DNA and RNA, have demonstrated incredible and broad usefulness in fields ranging from biomedicine to materials science. One very interesting application for synthetic nucleic acids is for something called gene silencing, where the expression of specific genes can be “turned off” by the addition of relatively short RNA strands.
Because many diseases are the result of overactive genes (e.g., cancer), developing an “off” switch could be very useful! Several obstacles, however, prevent widespread use of such therapy. First, the cost of synthesizing large amounts of RNA is very high. And just as importantly, the types of synthetic RNA that could be made until now breakdown very quickly or are quickly cleared away from the active site when used as a treatment in biological systems.
This present breakthrough developed from the discovery that a chemical reaction called “thiol-click” chemistry can be used to create nucleic acid polymers (where the “click” in the name comes from the fact that individual building blocks can be easily added onto each other, or clicked into place). Some advantages of this method include the low cost of starting materials, the fact that it is rapid, easily scalable, and that it can be designed as a single reaction.
Another key advantage is the fact that this method results in a non-natural sulfur-containing backbone, against which nucleic acid-degrading enzymes are ineffective. Thus, this strategy demonstrated significant progress in overcoming some of the major obstacles for biomedical application. However, the resulting polymer introduced a new obstacle intrinsic to its chemical structure. Whereas DNA and RNA are extremely water soluble, these new “Clickable Nucleic Acids” or “CNAs” were insoluble in water!
In order for CNAs to be viable for biological applications, they need to be water soluble, thus in this research, we changed their structure slightly. We started by adding a hydroxyl group onto the backbone, and then converted the hydroxyl to a highly hydrophilic sulfonate group. In this way, a new thymine-thymine monomer was synthesized with a sulfonate as a side group for every two thymines.
This monomer was polymerized to produce a thymine-CNA polymer that was water soluble! By simple changes to the reaction conditions, thymine-CNA polymers of different lengths were produced. Once CNA polymers had been created, we wanted to test if they could bind to a complementary strand of DNA. So thymine-CNA polymers were conjugated with silica and mixed with single stranded adenine DNA polymers conjugated with gold particles. Transmission electron microscope images revealed that the CNAs had indeed paired with the complementary DNA under these conditions.
Finally, another fascinating potential use of CNAs that we investigated was hydrogel production. Hydrogels have many uses including drug delivery, tissue engineering, etc. Because nucleic acids can be easily and specifically programmed to suit the needs of a given scenario, the use of CNA hydrogels could theoretically be very useful. To this end, we showed that thymine-CNA polymers could be combined with 8-arm PEG-Norbornene, a hydrogel backbone, and form CNA-PEG block copolymers. This approach provides a simple method for producing CNA hydrogels, i.e., by hybridizing with complementary multi-arm PEG-CNA chains.
The hydroxyl group in the backbone of the CNA polymers (i.e., before they are converted to sulfonate) could be used to couple other compounds of interest such as peptides or fluorescent probes, which would enable direct imaging of the nucleic acid analog. Meanwhile, we are focusing on precise control over the sequence (i.e., the ability to make non-repeating, predefined sequences) and molecular weight.
Research Article: Water-soluble Clickable Nucleic Acid (CNA) Polymer Synthesis by Functionalizing the Pendant Hydroxyl, Chemical Communications, 2017.