This invention relates to in vitro production of nucleic acids, particularly RNAs and specifically messenger RNAs (mRNA).

Provided herein are compositions and methods to assess the genomic landscape of fixed cells using light activated oligonucleotides that can be directed to the nucleus, mitochondria, or cytoplasm of fixed cells and that, upon activation, can be extended for in situ copying of nuclear single-stranded DNA (i.e., open chromatin), open mitochondrial DNA, and/or cytoplasmic RNA into barcoded complementary DNA. These methods also provide for gene specific 3D chromatin structural niche analysis.

The present invention provides, among other things, methods for large-scale production of a composition comprising full-length messenger RNA product that is substantially free of double-stranded RNA, and compositions produced using such methods and uses thereof. The present invention is based, in part, on the surprising discovery that mRNA product produced by in vitro transcription using an SP6 RNA polymerase is substantially free of double-stranded RNA. In one aspect, the present invention provides methods of generating large-scale mRNA product for mRNA therapy without need for a chromatography step.

The present invention relates to a novel variant RNA polymerase sigma factor 70 (δ.sup.70) polypeptide, a polynucleotide encoding the same, a microorganism containing the polypeptide, and a method for producing L-threonine by using the microorganism.

Disclosed herein is a pH activated nanoparticle that can be used to deliver labile therapeutic or diagnostic agents to the cytoplasm of cells. These nanoparticles allow the agents to escape the endosome by releasing a gas in an amount effective to disrupt the endosome and release the agents into the cytoplasm. The disclosed nanoparticles have a shell, such as a phospholipid bilayer shell, and a core containing a gas bound to a substrate by a pH sensitive interaction. Also disclosed herein is are methods for delivering a pH sensitive cargo to the cytoplasm of a cell, treating triple negative breast cancer (TNBC) in a subject, and treating HER2+ breast cancer in a subject.

Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Compositions and methods for synthesizing an RNA product are provided herein. For example, the present disclosure provides a method of producing an RNA product comprising incubating an in vitro transcription mixture, thereby producing an RNA product that comprises a plurality of single-stranded RNA molecules. In some embodiments, an in vitro transcription mixture comprises a DNA template comprising an RNA polymerase promoter sequence operatively linked to a target sequence; at least one RNA polymerase that recognizes the RNA polymerase promoter sequence; a plurality of ribonucleotides comprising at least two different types of ribonucleotides, each type comprising a different nucleoside; and a transcription buffer comprising an osmolyte.

Disclosed are compositions, systems, kits, and methods for detecting an analyte or target molecule in a sample by regulated in vitro transcription. The compositions, systems, kits, and methods typically comprise and/or utilize one or more components selected from: (a) an RNA polymerase; (b) an allosteric transcription factor (ATT), wherein the ATT binds an analyte or target molecule as a ligand; (c) an engineered transcription template; and/or any combination thereof. The engineered transcription template typically comprises a promoter sequence for the RNA polymerase and an operator sequence for the ATT. The promoter sequence and operator sequence are operably linked to a sequence encoding an RNA, wherein the ATT modulates transcription of the encoded RNA when the ATT binds the analyte or target molecule as a ligand, wherein the transcribed RNA generates a detectable signal in conjunction with a reporter molecule.

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

The present invention includes a method and kit for cDNA synthesis of a 3′UTR/poly(A) tail junction of cellular RNA comprising: obtaining RNA comprising a 3′UTR/poly(A) junction and a poly(a) tail; combining the RNA with three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP, dNTPs, and adaptor sequence-oligo-dT; performing reverse transcription of the RNA with a reverse transcriptase primed with the adaptor sequence-oligo-dT to form terminated cDNA fragments that are stochastically terminated upstream of the 3′UTR/poly(A) junction, but not within the poly(A) tail; isolating the terminated cDNA fragments; chemically ligating a functionalized 5′ adaptor to the terminated cDNA; and amplifying the chemically-ligated cDNA into an amplification product, wherein the cDNA is enriched for sequences at the 3′UTR/poly(A) tail junction without fragmentation or enzymatic ligation.