Generation of Cryptochrome-Targeting Nanobodies to Study Protein: Protein Interactions between Core Circadian Proteins

Presenter: John Joseph Ferriera

Faculty Sponsor: Michelle Farkas

School: UMass Amherst

Research Area: Biological Organisms

Session: Poster Session 6, 4:15 PM - 5:00 PM, Auditorium, A52

ABSTRACT

Circadian rhythms are autonomous, timekeeping mechanisms that oscillate on roughly 24-hour cycles. They regulate cellular processes like metabolism, DNA repair, and cell cycle progression. The mammalian molecular clock is controlled by a transcription-translation feedback loop involving four core clock proteins: BMAL1 and CLOCK, transcriptional activators, and CRY1/2 and PER1/2/3, transcriptional repressors. By day, BMAL1 and CLOCK dimerize and bind to E-box regulatory elements to promote CRY1/2 and PER1/2/3 transcription. By night, CRY and PER proteins dimerize and translocate into the nucleus, repressing their own transcription by interacting with CLOCK:BMAL1. While genetic methods have been used to study circadian proteins, they exhibit limitations like network compensation and redundancy. Small molecules have also been used to investigate protein function and modulate circadian rhythms, but they are limited in number, targets, and binding selectivity. Thus, novel molecular tools capable of selectively targeting core circadian protein associations are needed to understand better the roles of protein-protein interactions. We hypothesize that single-domain camelid antibodies, known as nanobodies, can specifically disrupt the CRY:PER repressor complex. Here, we describe candidate nanobody selection using in-house expressed and modified CRY1/2 proteins and screening methods, including magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). This selection is performed with a yeast nanobody display library. The affinity and selectivity of these nanobodies toward the PER2 binding site will be assessed using fluorescence polarization assays. In the future, we aim to evaluate the effects of these nanobodies in cellular models of circadian rhythms to further elucidate core clock protein roles.