Adenosine to inosine or A-to-I RNA editing executed by ADAR family proteins is abundant in the central nervous system (CNS). The availability of tools to monitor and manipulate both ADAR enzymes and A-to-I RNA modifications will facilitate investigation of the relationships between specific A-to-I conversions and their function in CNS development. The goal of this project is to develop a set of nanobodies to A-to-I RNA editing enzymes and their modified RNA products. These nanobodies are expected to serve not only as reagents for detection, quantification, or immunoprecipitation but also as intracellular antibodies capable of inhibiting the activity of target ADAR enzymes or binding to specific A-to-I modified RNAs in cells. As proof-of-principle, in Phase I, using Abzyme's proprietary self-diversifying single domain antibody library platform for high throughput antibody discovery, nanobodies to inosine-containing generic RNAs and site- specific A-to-I mRNA isoforms of mouse serotonin 2C receptor, as well as ADAR2-neutralizing nanobodies will be developed. In Phase II, nanobodies to mouse ADAR1 and ADAR3 as well as other CNS prominent A-to- I modified RNAs will be generated. In addition, transgenic knock-in mice will be generated with CNS-specific and expression-on-demand intrabodies to ADAR2, and the temporal impact of ADAR2 inactivation on CNS development will be investigated. The availability of such nanobodies will facilitate development of both immunoassays and cell lines or transgenic animals in which the function of ADAR enzymes or specific modified RNA can be investigated in time and space.
As a result of this proposed project, a set of nanobodies to A-to-I RNA modifications and ADAR enzymes will be generated. The availability of such tools will allow researchers to monitor and manipulate A-to- I modification processes and thereby investigate their roles in central nervous system development.