Nexstar Pharmaceuticals, Inc.

Somalogic, Incorporated

Gilead Sciences, Inc.

Sumedha D Jayasena

Larry M Gold

Dan W Drolet

Stanley C Gill

Steve Creighton

Gary P Kirschenheuter

Drew Smith

Dom Zichi

Greg Biesecker

Ken Davis

Nikos C Pagratis

Diane Tasset

Dan Nieuwlandt

Nebojsa Janjic

Chandra Vargeese

Dominic Zichi

Daniel W Drolet

Kenneth A Davis

Dominic A Zichi

Ross S Rabin

Daniel Drolet

Dan Drolet

A diagnostic biochip comprising a solid support to which one or more specific nucleic acid ligands is attached in a spatially defined manner is provided. Each nucleic acid ligand binds specifically and avidly to a particular target molecule contained within a test mixture, such as a bodily fluid. Also provided are methods for the preparation of nucleic acid ligand biochips. Further, methods for the use of the nucleic acid ligand biochip in diagnosis of a medical condition and quantitative detection of a target molecule are provided.

Nucleic acid ligand diagnostic biochip

A homogeneous assay that utilizes molecular beacons as the reporter and nucleic acid ligands as the sensor is described. This assay, called the ligand beacon assay, is for the detection of target molecules in a test mixture. The concept of the ligand beacon assay was tested using several proteins to which high affinity and specific nucleic acid ligands are available. The assay specifically detects the molecular target that binds the nucleic acid ligand with high affinity and specificity. The range of the assay is dictated by the concentration of the nucleic acid ligand/ligand beacon pair used in the assay. Target proteins were detected in buffer as well as in plasma, expanding its applicability to clinical use. This is a simple to use and fast assay format with the potential for automation for high throughput screening applications.

Homogeneous detection of a target through nucleic acid ligand-ligand beacon interaction

A Nucleic acid ligand &ldquo;Biochip&rdquo; is disclosed, consisting of a solid support to which one or more specific Nucleic acid ligands is attached in a spatially defined manner. Each Nucleic acid ligand binds specifically and avidly to a particular Target molecule contained within a Test mixture, such as a Bodily fluid. The Target molecules include, but are not limited to, proteins (cellular, viral, bacterial, etc.) hormones, sugars, metabolic byproducts, cofactor, and intermediates, drugs, and toxins. Contacting the Test mixture with the Biochip leads to the binding of a Target molecule to its cognate Nucleic acid ligand. Binding of Target to the Nucleic acid ligand results in a detectable change at each specific location on the Biochip. The detectable change can include, but is not limited to, a change in fluorescence, or a change in a physical parameter, such as electrical conductance or refractive index, at each location on the Biochip. The Biochip will then be read by a device, such as a fluorescence scanner or a surface plasmon resonance detector, that can measure the magnitude of the change at each location on the Biochip. The location of the change reveals what Target molecule has been detected, and the magnitude of the change indicates how much of it is present. The combination of these two pieces of information will yield diagnostic and prognostic medical information when signal patterns are compared with those obtained from Bodily fluids of individuals with diagnosed disorders (FIG. ). In principle, the Biochip could be used to test any chemically complex mixture provided that Nucleic acid ligands to components suspected of being present in the mixture are attached to the Biochip. Thus, the Nucleic acid ligand Biochip will have a wider use in environmental testing, etc.

A method is described for generating blended nucleic acid ligands containing non-nucleic acid functional units. Specifically, a SELEX identified RNA ligand to the integrin gpIIbIIIa is conjugated to the peptide Gly-Arg-Gly-Asp-Thr-Pro (SEQ ID NO:1). This blended RNA ligand inhibits the biological activity of gpIIbIIIa with high specificity. Also described is a single-stranded DNA ligand to elastase coupled to N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (SEQ ID NO:2). This elastase blended nucleic acid ligand inhibits the biological activity of elastase.

Systematic evolution of ligands by exponential enrichment: blended selex

A Nucleic acid ligand &ldquo;Biochip&rdquo; is disclosed, consisting of a solid support to which one or more specific Nucleic acid ligands is attached in a spatially defined manner. Each Nucleic acid ligand binds specifically and avidly to a particular Target molecule contained within a Test mixture, such as a Bodily fluid. The Target molecules include, but are not limited to, proteins (cellular, viral, bacterial, etc.) hormones, sugars, metabolic byproducts, cofactor, and intermediates, drugs, and toxins. In principle, the Biochip could be used to test any chemically complex mixture provided that Nucleic acid ligands to components suspected of being present in the mixture are attached to the Biochip. Thus, the Nucleic acid ligand Biochip will have a wider use in environmental testing, etc.

A method for detecting a target molecule in a test mixture suspected of containing said target molecule is described. The nucleic acid ligand capable of binding to the target molecule has a first sequence A and a second sequence B, which are partially complementary sequences that form an imperfect intramolecular duplex, which unwinds upon the binding of the target to the nucleic acid ligand. Sequences A and B are able to participate in extramolecular hybridization reactions only when the duplex is unwound. Three different cascade nucleic acids contain a first sequence and a second sequence, which are partially complementary sequences. At least one sequence is exactly complementary to A or B. The second sequence may be complementary to A or B, or may be a third sequence C, or its complement. The test mixture suspected of containing the target molecule is contacted with the nucleic acid ligand, causing the duplex of the nucleic acid ligand to unwind such that sequences A and B become available for extramolecular hybridization. This mixture is contacted with the first, second, and third cascade nucleic acids so that the unpaired A and B sequences triggers a cascade of intermolecular hybridization involving the cascade nucleic acids in which intermolecular hybridization takes place between A and its complement, B and its complement, and between C its complement, leading to the formation of a multimolecular hybridization complex. The presence of the multimolecular hybridization complex is then detected.

A Nucleic acid ligand &ldquo;Biochip&rdquo; is disclosed, consisting of a solid support to which one or more specific Nucleic acid ligands is attached in a spatially defined manner. Each Nucleic acid ligand binds specifically and avidly to a particular Target molecule contained within a Test mixture, such as a Bodily fluid The Target molecules include, but are not limited to, proteins (cellular, viral, bacterial, etc.) hormones, sugars, metabolic byproducts, cofactor, and intermediates, drugs, and toxins. Contacting the Test mixture with the Biochip leads to the binding of a Target molecule to its cognate Nucleic acid ligand. Binding of Target to the Nucleic acid ligand results in a detectable change at each specific location on the Biochip. The detectable change can include, but is not limited to, a change in fluorescence, or a change in a physical parameter, such as electrical conductance or refractive index, at each location on the Biochip. The Biochip will then be read by a device, such as a fluorescence scanner or a surface plasma resonance detector, that can measure the magnitude of the change at each location on the Biochip. The location of the change reveals what Target molecule has been detected, and the magnitude of the change indicates how much of it is present The combination of these two pieces of information will yield diagnostic and prognostic medical information when signal patterns are compared with those obtained from Bodily fluids of individuals with diagnosed disorders FIG.,). In principle, the Biochip could be used to test any chemically complex mixture provided that Nucleic acid ligands to components suspected of being present in the mixture are attached to the Biochip. Thus, the Nucleic acid ligand Biochip will have a wider use in environmental testing, etc.

Nucleic acid ligand diagnostic Biochip

This invention discloses high-affinity oligonucleotide ligands to the thermostable Taq polymerase, Tth polymerase and TZ05 polymerase. Specifically, this invention discloses DNA ligands having the ability to bind to the Taq, Tth and TZ05 polymerases and the methods for obtaining such ligands. The ligands are capable of inhibiting polymerases at any predetermined temperature.

Nucleic acid ligand inhibitors to DNA polymerases

The presence of a target in a test mixture or the concentration of such target can be determined by a method relying on fluorescence emission measurement. Such method utilizes a nucleic acid ligand to the target and a ligand beacon comprising: i) a nucleic acid sequence complimentary to at least a portion of said nucleic acid ligand, ii) a fluorescent group, and iii) a fluorescence-modifying group. The emission profile of the ligand beacon is altered based on the presence and/or concentration change of the target in the test mixture.

A method is described for generating blended nucleic acid ligands containing non-nucleic acid functional units. Specifically, a SELEX identified RNA ligand to the integrin gpIIbIIIa is conjugated to the peptide Gly-Arg-Gly-Asp-Thr-Pro (SEQ ID NO:1). This blended RNA ligand inhibits the biological activity of gpIIbIIIa with high specificity. Also described is a single-stranded DNA ligand to elastase coupled to N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl (SEQ ID NO:3) ketone. This elastase blended nucleic acid ligand inhibits the biological activity of elastase.

Systematic evolution of ligands exponential enrichment: blended selex

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to cytokines. Included in the invention are specific nucleic acid ligands to IFN-gamma, IL-4, IL-10, TNF-alpha, and RANTES.

High affinity nucleic acid ligands of cytokines

This invention pertains to methods of determining the presence and/or quantity of homocysteine in a sample containing other thiol-containing compounds. The methods of this invention involve modifying the homocysteine to facilitate the separation of homocysteine from cysteine. An assay protocol comprises adjusting the conditions of the sample suspected of containing homocysteine so that homocysteine forms homocysteine thiolactone, separating the homocysteine thiolactone from free thiol-containing compounds present in the sample, including cysteine, reconverting the homocysteine thiolactone to homocysteine, and determining the presence and/or quantity of homocysteine in the sample by conventional means.

Homocysteine assay

Homogeneous detection of a target through nucleic acid ligand-ligand

This invention discloses novel detection methods for determining the presence of a target compound in a substance using nucleic acid ligands as detection molecules. Specifically, the substance is bound to a solid support matrix, such as those used in blot procedures, and detection of the target molecule is accomplished using the affinity and specificity of nucleic acid ligands to the target molecule. The method utilized herein for identifying and preparing said nucleic acid ligands is called SELEX. The method of the present invention is additionally useful to isolate the target compounds from the various substances.

Growing community of inventors

Boulder, CO, United States of America

Sumedha D Jayasena