# US 5,705,344 A · High-throughput screening assay for inhibitors of nucleic acid helicases

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- Number: US 5,705,344 A
- Document ID: US5705344A
- Status: Granted
- Kind: A
- Application number: 08/616,046
- Filed: 1996-03-14
- Granted: 1998-01-06
- Earliest claimed: 1996-03-14
- Assignees: Tularik Inc.
- Inventors: Heidi Giordano; Michael G. Peterson; Mohanram Sivaraja
- Web page: https://patentgrep.com/patent/US5705344A

## CPC

- C12Q 1/68: involving nucleic acids
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides screening assays for identifying an inhibitor of a pathogenic helicase activity, e.g. a helicase derived from a pathogenic infectious organism such as a bacterium, protozoan or fungus. Helicase activity is detected by the solid-phase, preferential capture of retained (non-liberated) single-stranded nucleic acid comprising a detectable label.

## Claims

### Claim 1

A method of identifying an agent that modulates the activity of a helicase comprising the following steps in consecutive order:
a) forming a mixture of a labeled first nucleic acid hybridized to a complementary unlabeled second nucleic acid, a helicase, a nucleoside triphosphate, and a candidate agent, wherein said first and second nucleic acids are both DNA or are both RNA;
b) incubating said mixture in a reservoir under conditions wherein said helicase would unhybridize said first and second nucleic acids in the absence of said agent;
c) immobilizing said second nucleic acid on a solid substrate by direct noncovalent binding, wherein said second nucleic acid has a greater affinity for said solid substrate than does the first nucleic acid;
d) separating any unhybridized labeled first nucleic acids to obtain isolated, immobilized second nucleic acids;
e) measuring the amount of said label retained on the immobilized second nucleic acid, wherein the amount of said label is indicative of the modulation of helicase activity by said agent.

### Claim 2 (depends on 1)

The method of claim 1, wherein said solid substrate is a micro-bead and said greater affinity results from said second nucleic acid being of greater length than said first nucleic acid.

### Claim 3 (depends on 1)

The method of claim 1, wherein said second nucleic acid comprises a ligand, wherein said greater affinity results from said ligand having a greater affinity for said solid substrate than does said first nucleic acid.

### Claim 4 (depends on 3)

The method of claim 3, wherein said solid substrate is coated with avidin, said ligand is biotin and said label is other than biotin.

### Claim 5 (depends on 3)

The method of claim 3, wherein said solid substrate is coated with a digoxigenin-specific antibody, said ligand is digoxigenin and said label is other than digoxigenin.

### Claim 6 (depends on 1)

The method of claim 1, wherein said label is a radiolabel.

### Claim 7 (depends on 1)

The method of claim 1, wherein said label is a fluorescent label.

### Claim 8 (depends on 3)

The method of claim 3, wherein said label is biotin and said ligand is other than biotin.

### Claim 9 (depends on 3)

The method of claim 3, wherein said label is digoxigenin and said ligand is other than digoxigenin.

### Claim 10 (depends on 1)

The method of claim 1, wherein said separating step comprises filtering said solid substrate.

### Claim 11 (depends on 1)

The method of claim 1, wherein said separating step is performed in a tube having a fluid passage comprising a reservoir portion and a evacuation portion a filter extending transversely across said passage and separating said reservoir portion from said evacuation portion, said filter being changeable from water-impermeable to water-permeable having a maximam pore size sufficiently large to freely pass said any unhybridized first nucleic acid and sufficiently small to retain said solid substrate.

### Claim 12 (depends on 10)

The method of claim 10, wherein said filter comprises a first membrane being changeable from water-impermeable to water-permeable by contacting said first membrane with an effective concentration of an organic solvent and a second membrane being water-permeable and having a maximam pore size less than about 200 um diameter.

### Claim 13 (depends on 1)

The method of claim 1, wherein said helicase is a pathogenic helicase.

### Claim 14 (depends on 1)

The method of claim 1, wherein said hellcase is a pathogenic fungal helicase.

### Claim 15 (depends on 1)

The method of claim 1, wherein said helicase is a pathogenic human helicase.

### Claim 16 (depends on 1)

The method of claim 1, wherein said helicase is a pathogenic bacterial helicase.

### Claim 17 (depends on 1)

The method of claim 1, wherein said helicase is a pathogenic viral helicase.