Triazole linkages and backbone branches in nucleic acids for biological and extra-biological applications.

摘要

The recently increasing evidence of nucleic acids' alternative roles in biology and potential as useful nanomaterials and therapeutic agents has enabled the development of useful probes, elaborate nanostructures and therapeutic effectors based on nucleic acids. The study of alternative nucleic acid structure and function, particularly RNA, hinges on the ability to introduce site-specific modifications that either provide clues to the nucleic acid structure function relationship or alter the nucleic acid's function. Although the available chemistries allow for the conjugation of useful labels and molecules, their limitations lie in their tedious conjugation conditions or the lability of the installed probes. The development and optimization of click chemistry with RNA now provides the access to a robust and orthogonal conjugation methodology while providing stable conjugates. Our ability to introduce click reactive groups enzymatically, rather than only in the solid-phase, allows for the modification of larger, more cell relevant RNAs. Additionally, ligation of modified RNAs with larger RNA constructs through click chemistry represents an improvement over traditional ligation techniques. We determined that the triazole linkage generated through click chemistry is compatible in diverse nucleic acid based biological systems.;Click chemistry has also been developed for extra-biological applications, particularly with DNA. We have expanded its use to generate useful polymer-DNA conjugates which can form controllable soft nanoparticles which take advantage of DNA's properties, i.e. DNA hybridization and computing. Additionally, we have generated protein-DNA conjugates and assembled protein-polymer hybrids mediated by DNA hybridization. The use of click chemistry in these reactions allows for the facile synthesis of these unnatural conjugates. We have also developed backbone branched DNA through click chemistry and showed that these branched DNAs are useful in generating well-defined architectures based solely on DNA.;While backbone branched DNAs are useful for nanotechnological applications, backbone branches in RNA occur in nature and are involved in the distinct but related processes of splicing, debranching and RNAi. Therefore we have developed protocols for the synthesis of backbone branched nucleic acids in the solid-phase using photoprotecting groups. Using the synthesized backbone branched RNAs we have uncovered a specific substrate requirement of debranching enzyme which distinguishes it from other homologous proteins with alternative functions.;Finally, through the marriage of click chemistry and backbone branches, we have produced useful progeny in the synthesis of lariat RNAs. We investigated the potential of these lariats as therapeutic agents by synthesizing siRNA sequences as lariats. We showed that these lariats are efficiently debranched by debranching enzyme and are able to induce an RNAi response in vivo. Altogether, the development of click chemistry and backbone branched nucleic acids represents a significant advantage in the ability to modify nucleic acid structure and affect its function. I envision that these methods can become generally useful to probe nucleic acid systems, useful nanomaterials and functional effectors in nucleic acid based therapies.