Patent ID: 11959121
Assignee: MAX-PLANCK-GESELLSCHAFT ZUR FÖRDERUNG DER WISSENSCHAFTEN E. V.
Field: Measurement (Instruments)
Classification: CPC C  G | IPC C  G

Claim 0:
1. A sensor molecule for the bioluminescence resonance energy transfer (BRET)-based detection of reduced nicotinamide adenine dinucleotide phosphate (NADPH), the sensor comprising a segment A connected via a linker to a segment B, wherein each of segment A and segment B comprises a member of a BRET pair comprising a donor moiety and an acceptor moiety,
further characterized in that
(i) segment A comprises a binding protein (BP) for NADPH, the BP being dihydrofolate reductase (DHFR; EC 1.5.1.3) of E. coli (UniprotKB accession number POABQ4) having the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 95% sequence identity to SEQ ID NO:2, or a functional homolog thereof selected from SEQ ID NOS:3-6 and variants thereof having at least 95% sequence identity to any of SEQ ID NOS:3-6, wherein the BP is capable of binding NADPH, and wherein the BP comprises a heterologous protein domain inserted at or replacing at least part of the region corresponding to positions 22 to 25 of E. coli DHFR, said heterologous protein domain comprising as one member of the BRET pair a luciferase enzyme as bioluminescent donor protein (BDP);
(ii) segment B comprises a ligand (L) capable of intramolecular binding to said BP only in the presence of NADPH and wherein segment B comprises a fluorescence acceptor moiety whose excitation spectrum at least partially overlaps with an emission spectrum of the luciferase;
wherein the donor moiety and the acceptor moiety are in a suitable juxtaposition to yield a BRET signal when L is bound to BP, and wherein NADPH-induced binding of L to BP results in an increase in BRET efficiency.