SUMMARY: Chemistry-driven strategies that allow proteins to be manipulated in ways not permitted by standard genetics are now indispensable tools in both basic and applied aspects of biomedical science. Of the various platform technologies in common use, those that rely on manipulation of intein-mediated protein splicing have proven especially powerful owing to broad compatibility with both in vitro and in vivo applications. Work performed during the current funding period has led to the discovery, characterization and eventual engineering of several inteins, especially those split into two mutually-dependent fragments that support protein trans-splicing upon complexation. We have employed these ‘protein ligases’ to solve a number of biochemical problems, primarily in the chromatin biology area. In the next phase of this program, we will exploit recent advances in our understanding of protein trans-splicing, to develop split intein tools designed to address a series of problems also in the area of epigenetics. Accordingly, a concerted protein ‘transposition’ reaction will be developed that allows replacement of internal regions of intact target proteins with synthetic cassettes containing chemical probes such as crosslinkers and photosensitizers (Aim 1). This cut-and paste system will be used to define and manipulate epigenetic complexes involved in higher-order chromatin structure. We will also develop various controllable versions of split inteins for use in both basic science and synthetic biology applications. In one line of enquiry (Aim 2), we will use these tools to rapidly generate oncofusion proteins such AML-ETO, thereby allowing characterization of the early stages of cell transformation. Lastly, in Aim 3, we will develop protein ‘actuators’ that use conditional protein splicing to convert a biochemical input of choice into a user-defined biochemical output. These ‘actuators’ will form the basis of epigenetic circuits that are dependent on Boolean logic operations and that allow higher- order chromatin complexes to be monitored in cells. While our focus is in the epigenetic area, we imagine that the technologies developed in the context of this work will expand the type of protein systems, and hence biological problems, accessible to interrogation through chemical biology strategies.