Atp-independent bioluminescent reporter variants to improve in vivo imaging

An engineered luciferase protein of SEQ ID NO:

• 46 (BREP), SEQ ID NO:
• 47 (BRIPO0.5), or SEQ ID NO:
• 48 (BRIPO) or a protein having 95% homology with a protein of SEQ ID NO:
• 46 (BREP), SEQ ID NO:
• 47 (BRIPO0.5), or SEQ ID NO:
• 48 (BRIPO).

A method of producing a bioluminescent signal comprising contacting a monopotassium salt of a bioluminescent indicator of the formula

• with an engineered luciferase protein of SEQ ID NO:
• 1 (BREP), SEQ ID NO:
• 2 (BRIPO0.5), or SEQ ID NO:
• 3 (BRIPO) or a protein having 95% homology with a protein of SEQ ID NO:
• 46 (BREP), SEQ ID NO:
• 47 (BRIPO0.5), or SEQ ID NO:
• 48 (BRIPO).

A bioluminescent calcium indicator composition comprising:

• (a) a genetically engineered luciferase fusion protein comprising a NanoLuc-derived luciferase and a red fluorescent protein with at least 80% or more sequence identity to the proteins with SEQ. ID. NO:
• 49 or SEQ. ID. NO:
• 50, wherein the luciferase fusion protein is engineered to include a calcium-binding domain that modulates bioluminescence in response to calcium ion concentration;
• and,
• (b) a luciferin substrate;
• wherein, upon binding of calcium ions, the indicator exhibits an increase in red-shifted bioluminescence emission suitable for in vivo imaging of calcium dynamics in mammalian cells or tissues.

A compound, comprising:

• a coelenterazine (CTZ) or diphenylterazine (DTZ) analog, covalently conjugated at the C3 carbonyl group to a cleavable substituent;
• wherein said substituent is selected from:
• hydrophilic polymers, saccharides, amino acids, peptides, zwitterionic groups, or combinations thereof;
• wherein the substituent enhances aqueous solubility, stability, pharmacokinetics, or in vivo distribution of the luciferin;
• and,
• wherein the conjugate is cleavable in vivo to release the luciferin.

A compound, comprising:

• a coelenterazine (CTZ) or diphenylterazine (DTZ) analog, covalently conjugated at the C3 carbonyl group to a polyethylene glycol (PEG) moiety;
• wherein, said PEG has a molecular weight between 2 kDa and 20 kDa;
• and,
• wherein the conjugate is cleavable in vivo to release the luciferin.