Leland Stanford Junior University

Discoverx Corporation

Joseph L Horecka

Ronald W Davis

Pyare L Khanna

Ulrich Schlecht

Robert P St Onge

Sasha F Levy

Peter Fung

Richard M Eglen

Roxana Jalili

Henrik H J Persson

Henrik HJ Persson

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

Highly specific circular proximity ligation assay

A novel method for preparing sequence-verified oligonucleotides is disclosed. In particular, the invention relates to a simple, affordable, and scalable method that combines high-throughput mating of yeast clones, a unique selectable system for combining DNA sequences in yeast, and next-generation sequencing. This method allows sequence-verified oligonucleotides to be readily isolated from complex libraries.

Scalable method for isolation and sequence-verification of oligonucleotides from complex libraries

Improved methods of determining the intracellular state of a protein as well as modifications of the protein are provided by introducing a surrogate fusion protein comprising a member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with the surrogate fusion protein to a change in environment, e.g. a candidate drug, the cells are lysed, the lysate separated into fractions or bands, conveniently by gel electrophoresis and transferring the proteins by Western blot to a membrane. The bands on the membrane are developed using the other member of the enzyme fragment complementation complex and a substrate providing a detectable signal. The method is found to provide high sensitivity and the ability to observe modifications of the target protein.

Analysis of intracellular modifications

Methods and reagents are provided for measuring protease activity. The reagent comprises a surface to which is linked an enzyme donor fragment through a protease recognition sequence, where the enzyme donor fragment complexes with an enzyme acceptor fragment to form an active indicator enzyme when the enzyme donor fragment is cleaved from said surface. Conveniently, the surface is a cell membrane surface where the reagent is expressed in the cell. The method comprises bringing together the reagent, the protease, the enzyme acceptor and substrate for the indicator enzyme and measuring the indicator enzyme activity as a measure of the protease activity. The method finds application in screening for compounds modulating the activity of the protease.

Protease EFC cell surface fusion protein assay

A protein reagent is provided for measuring protease enzyme activity in a sample. The protein reagent comprises two inhibition entities joined by a linker comprised of an indicator enzyme donor and an amino acid sequence susceptible to enzymatic cleavage. The protein reagent is substantially inhibited from binding to the cognate enzyme acceptor fragment, while the product of the enzymatic cleavage binds to the cognate enzyme acceptor fragment to form a functional indicator enzyme. The indicator enzyme activity is related to the protease enzyme activity of the sample.

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Stanford, CA, United States of America

Joseph L Horecka