New England Biolabs, Inc.

George Tzertzinis

Jennifer Ong

Shaorong Chong

Vladimir Potapov

Haruichi Asahara

Kuo-Chan Hung

Ivan R Correa, Jr

Laurence Ettwiller

Ira Schildkraut

Madalee G Wulf

Yinhua Zhang

Christopher J Noren

Nan Dai

Larry A McReynolds

George R Feehery

Bo Yan

Sanjay Kumar

John Buswell

Corinna Tuckey

Claude V Maina

A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.

Thermostable variants of T7 RNA polymerase

A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5' end of 5&#x2032;-diphosphorylated or 5&#x2032;-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5&#x2032; or 3&#x2032; end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.

Methods for labeling a population of RNA molecules

Provided herein is a method for making an cDNA library, comprising adding an affinity tag-labeled GMP to the 5' end of targeted RNA species in a sample by optionally decapping followed by incubating the sample with an affinity tag-labeled GTP and a capping enzyme, enriching for RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag, reverse transcribing the enriched RNA to produce a population of cDNAs, and adding a tail to the 3&#x2032; end of the population of cDNAs using a terminal transferase, to produce an cDNA library.

Enrichment and sequencing of RNA species

A method for obtaining a mixture of heterogenous short double-stranded RNA molecules suitable for use in gene silencing (hsiRNA) by subjecting large double-stranded RNA to enzymatic cleavage under specified conditions. The resulting mixture consistently includes enhanced representation of fragments having a size of 21-22 nucleotides absent any fractionation step. The fragments contain sequences that collectively span the entire length of the large double-stranded RNA from which they are derived. Double-stranded RNA with sequences that individually represent segments of a target mRNA may be analyzed using the methods described herein to identify the most active subset of hsiRNA fragments or individual siRNA fragments for achieving gene silencing for any gene or transcribed sequences. A method is additionally provided for preparing and cloning DNA encoding selected siRNA, hsiRNA mixtures or hairpin sequences to provide a continuous supply of a gene silencing reagent derived from any long double-stranded RNA.

Methods and compositions relating to gene silencing

Compositions and methods are provided for preparing an hsiRNA mixture and for silencing of gene expression in vivo. The composition relates to a mutant RnaseIII. The methods are directed to reacting a preparation of dsRNA with an effective amount of a mutant RNAse III to produce the hsiRNA mixture.

Compositions and methods for generating short double-stranded RNA using mutated RNAse III

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Ipswich, MA, United States of America

George Tzertzinis