Abstract: Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.

Abstract: Methods and compositions for capping RNA in an in vitro transcription mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.

Abstract: Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.

Abstract: Providing herein, among other things, are kits, compositions and methods that relate to DNA fragmentation. An embodiment of a composition provides combining: one or more enzymes capable of nick translating activity, a dNTP mix comprising at least one dNTP having a modified base, and at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if its recognition site contains the modified base. When the composition is added to a sample comprising a double-stranded DNA template that comprises recognition sites for the modification-sensitive nicking endonuclease, a reaction mix was produced which could be incubated for any time period in excess of about 5 minutes to produce fragments of a desired size of the double-stranded DNA template. In this method, the fragments produced include the modified base and, as such, are not re-nicked by the nicking endonuclease.

Abstract: Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.

Abstract: Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.

Abstract: Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: Provided herein is a thermolabile proteinase and methods of using the same. In some embodiments, the thermolabile proteinase may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs:1-11 and at least one amino acid substitution in helix 3. The thermolabile proteinase is active at a temperature in the range of 4° C.-40° C. and is inactivated by raising the temperature to above 50° C., where the proteinase is substantially inactive at 65° C.

Abstract: Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting different template sequences in a single nucleic acid target. The kits and methods also include guanidium salts that enhance the sensitivity of the assay.

Abstract: Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.

Abstract: Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

Abstract: Provided herein is a method for isolating high molecular weight (HMW) DNA using beads that are at least 200 ?m in diameter that utilizes a device for retaining the beads and where the purified DNA eluant exits the device without shearing the HMW DNA. In some embodiments, the method comprises precipitating the DNA onto the beads, washing the beads in the device, and then eluting the DNA from the beads therein while substantially avoiding shear. Compositions and kits for practicing the method are also provided.

Abstract: Disclosed herein are isolated peptides inhibit activity of a cofactor-independent phosphoglycerate mutase. In some examples, the isolated peptide is 6-20 amino acids long and includes the amino acid sequence of any one of SEQ ID NOs: 1-22 or 54, an analog or derivative thereof, or a pharmaceutically acceptable salt or ester thereof. In some examples, the peptide is a cyclic peptide with an N-terminal ring of 6-15 amino acids (for example, 6-10 amino acids) and a C-terminal linear portion of 1-9 amino acids (for example, 3-8 amino acids. Also disclosed h are methods of treating or inhibiting an infection in a subject, including administering to the subject an effective amount of a composition including one of more of the disclosed peptides, or analogs or derivative thereof, or pharmaceutically acceptable salts or esters thereof.

Abstract: Mutant bacteriophage DNA ligases that have increased tolerance to salt and/or heat is provided. Methods, compositions and kits that employ the same are also provided.

Abstract: Compositions are provided for 3? adapters and methods of use are provided that include methods requiring a plurality of ligation steps involving a single-stranded target polynucleotide and 3? and 5? adapters. Embodiments of the 3? adapters comprise a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3? adapters, the single-stranded region is released by cleaving the cleavable linker.

Abstract: Disclosed herein are isolated peptides inhibit activity of a cofactor-independent phosphoglycerate mutase. In some examples, the isolated peptide is 6-20 amino acids long and includes the amino acid sequence of any one of SEQ ID NOs: 1-22 or 54, an analog or derivative thereof, or a pharmaceutically acceptable salt or ester thereof. In some examples, the peptide is a cyclic peptide with an N-terminal ring of 6-15 amino acids (for example, 6-10 amino acids) and a C-terminal linear portion of 1-9 amino acids (for example, 3-8 amino acids. Also disclosed h are methods of treating or inhibiting an infection in a subject, including administering to the subject an effective amount of a composition including one of more of the disclosed peptides, or analogs or derivative thereof, or pharmaceutically acceptable salts or esters thereof.

Abstract: Providing herein, among other things, is a method comprising incubating a double-stranded nucleic acid having a nick with a nick translating activity, a ligase, and a nucleotide mix comprising at least one modified nucleotide, to generate a product comprising a patch of a newly synthesized strand of a duplex nucleic acid containing a plurality of modified nucleoside monophosphates that are at or adjacent to the site of the nick. In some embodiments, the method may be used to map damaged nucleoside monophosphates in a nucleic acid. Compositions and kits for use in performing the method are also provided.