New England Biolabs, Inc.

Larry A McReynolds

Thomas C Evans, Jr

Gregory Lohman

Daniela Munafo

Yinhua Zhang

Christopher J Noren

George Tzertzinis

George R Feehery

Jingmin Jin

Corinna Tuckey

Compositions and methods are provided for ligating polynucleotides having a length that is greater than 8 nucleotides on an RNA splint. The ligation reaction provides consistent results in high or low ATP concentrations. The reaction can occur rapidly and is generally at least 10 fold more efficient than T4DNA ligase under optimal conditions for T4DNA ligase and the reaction time is less than 6 hours for example, less than 1 hour.

Ligase activity

Methods are provided for ligating a 3' adapter and a 5&#x2032; adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3&#x2032; adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3&#x2032; adapter and the ligated 3&#x2032; adapter. Subsequently, a 5&#x2032; adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.

Method for reducing adapter-dimer formation

Methods and compositions are provided for detecting small target RNAs where the target RNA may be single-stranded or double-stranded and may be contained in a mixture of RNAs of different types and sizes. The methods and compositions utilize a p19 fusion protein that is capable of binding double-stranded RNA in a size-specific but sequence-independent manner and is further capable of binding to a matrix such as beads or plastic microwell plates. By labeling the p19 fusion protein or the target RNA in a polynucleotide duplex either directly or indirectly, low levels of target RNA including microRNAs can be detected from cells. This can be applied to diagnosis of pathological conditions.

Methods and compositions for detection and enrichment of target small RNAs

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.

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Beverly, MA, United States of America

Larry A McReynolds