The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.

A polynucleotide encoding a modified luciferase polypeptide. The modified luciferase polypeptide has at least 60% amino acid sequence identity to a wild-type Oplophorus luciferase and includes at least one amino acid substitution at a position corresponding to an amino acid in a wild-type Oplophorus luciferase of SEQ ID NO:1. The modified luciferase polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the wild-type Oplophorus luciferase.

Luciferases which are different from those known heretofore have been desired. A luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from the group consisting of valine at the position of 44, alanine at the position of 54 and tyrosine at the position of 138 is substituted with other amino acid(s) in the amino acid sequence of SEQ ID NO: 2.

Described herein are block copolymers, and methods of making and utilizing such copolymers. The described block copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the block copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the block copolymer is endosomolytic and capable of delivering an oligonucleotide (e.g., an mRNA) to a cell. Compositions comprising a block copolymer and an oligonucleotide (e.g., an mRNA) are also disclosed.

The present invention provides a method for providing modified mRNAs of reduced immunogenicity and/or immunostimulatory capacity for use in protein replacement therapy. The invention further provides modified mRNAs and pharmaceutical compositions comprising the modified mRNAs according to the invention for use in protein replacement therapy.

Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)-based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the G subunit and a G interacting protein ( IP) for the binding to the G dimer. These biosensors comprises (1) a IP and (2) a G or G protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.

An isolated polynucleotide encoding a modified luciferase polypeptide and substrates. The OgLuc variant polypeptide has at least 60% amino acid sequence identity to SEQ ID NO: 1 and at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID NO: 1. The OgLuc variant polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the corresponding polypeptide of the wild-type Oplophorus luciferase.

Provided herein are systems and methods for the detection of an analyte or analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and/or quantifying a target analyte using a bioluminescent complex comprising substrates, peptides, and/or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.

The present invention provides a mutant beetle luciferase and the like, having mutation in which the amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type Photinus pyralis luciferase is isoleucine, leucine or phenylalanine, mutation in which the amino acid corresponding to leucine at position 376 in the aforementioned sequence is proline, mutation in which the amino acid corresponding to glutamic acid at position 455 in the aforementioned sequence is valine, alanine, serine, leucine, isoleucine or phenylalanine, or mutation in which the amino acid corresponding to glutamic acid at position 488 in the aforementioned sequence is valine, alanine, serine, leucine, isoleucine or phenylalanine, in the amino acid sequence encoding a wild-type beetle luciferase, and characterized in that a luminescence intensity due to a luciferin-luciferase luminescence reaction in a 0.9% by mass NaCl solution is 50% or more of that in a NaCl-free solution.

Luciferin-containing substrates are provided including a substrate and luciferin dried on the substrate. The luciferin-containing substrate is free of a detectable amount of reactive luciferase. The substrate is optionally a nonwoven substrate. Monitoring devices are also provided. The monitoring devices include a test element having a test portion, a detection reagent comprising luciferase, a luciferin-containing substrate, and a container having a first end with an opening and a second end opposite the first end. The container is configured to receive the test portion and configured to be operationally coupled to an analytical instrument. The detection reagent and the luciferin each are capable of participating in one or more chemical reaction that results in the formation of a detectable product.