Abstract“Molecular glue” (MG) is a term coined to describe the mechanism of action of the plant hormone auxin and subsequently used to characterize synthetic small molecule protein degraders exemplified by immune-modulatory imide drugs (IMiDs). Prospective development of MGs, however, has been hampered by its elusive definition and thermodynamic characteristics. Here, we report the crystal structure of a dual-nanobody cannabidiol-sensing system, in which the ligand promotes protein-protein interaction in a manner analogous to auxin. Through quantitative analyses, we draw close parallels among the dual-nanobody cannabidiol sensor, the auxin perception complex, and the IMiDs-bound CRL4CRBN E3, which can bind and ubiquitinate “neo-substrates”. All three systems, including the recruitment of IKZF1 and CK1α to CRBN, are characterized by the lack of ligand binding activity in at least one protein partner and an under-appreciated preexisting low micromolar affinity between the two proteinaceous subunits that is enhanced by the ligand to reach the nanomolar range. These two unifying features define MGs as a special class of proximity inducers distinct from bifunctional compounds and can be used as criteria to guide target selection for future rational discovery of MGs.
Chemically induced dimerization (CID) systems, in which two proteins
dimerize only in the presence of a small molecule ligand, offer versatile tools
for small molecule sensing and actuation. However, only a handful of CID systems
exist and creating one with the desired sensitivity and specificity for any
given ligand is an unsolved problem. Here, we developed a
combinatorial
binders-enabled
selection of CID (COMBINES-CID) method broadly
applicable to different ligands. We demonstrated a proof-of-principle by
generating nanobody-based heterodimerization systems induced by cannabidiol with
high ligand selectivity. We applied the CID system to a sensitive sandwich
ELISA-like assay of cannabidiol in body fluids with a detection limit of
~0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution
for expanding the biosensor toolkit for small molecule detection.
Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays for wide implementation at a high frequency. This paper presents a design for an in-solution assay pipeline, featuring
nano
body-functionalized
nano
particles for
r
apid,
e
lectronic
d
etection (Nano2RED) of Ebola and COVID-19 antigens. Synthetic nanobody binders with high affinity, specificity, and stability are selected from a combinatorial library and site-specifically conjugated to gold nanoparticles (AuNPs). Without requiring any fluorescent labelling, washing, or enzymatic amplification, these multivalent AuNP sensors reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or portable electronic circuitry. With Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (the limit of detection of ∼10 picogram (pg)/mL, or 0.13 13 picomolar (pM) for sGP and ∼40 pg/mL, or ∼1.3 pM for RBD in diluted human serum), a high specificity, a large dynamic range (∼7 logs),and fast readout within minutes. The rapid detection, low material cost (estimated <$0.01 per test), inexpensive and portable readout system (estimated <$5), and digital data output, make Nano2RED a particularly accessible assay in screening of patient samples towards successful control of infectious diseases.
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