Polynucleotide assay reagent and method

• US 5,217,866 A
• Filed: 12/18/1986
• Issued: 06/08/1993
• Est. Priority Date: 03/15/1985
• Status: Expired due to Term

• Current Assignee: Neu-Gene Development, AntiVirals, Inc. (Sarepta Therapeutics, Inc.)
• Original Assignee: Anti-Gene Development Group
• Inventors: Summerton, James, Weller, Dwight
• Primary Examiner(s): YARBROUGH, AMELIA
• Application Number:
  US06/944,707
• Time in Patent Office: 2,364 Days
• Field of Search: 435/6, 435/91, 435/172.3, 435/968, 544/118, 544/122, 544/123, 528/391, 528/403, 528/405, 528/406, 436/501, 436/518, 436/530, 436/531, 436/800, 436/56, 935/78, 935/77
• US Class Current: 435/6.12
• CPC Class Codes: A61K 47/545 Heterocyclic compounds A61K47/558 takes precedence
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/59 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification

Abstract

A diagnostic reagent and system for determination of a single-stranded polynucleotide analyte having a selected target sequence. The reagent includes a solid support and multiple support-bound polymers designed to bind specifically to the analyte. Each polymer is composed of a sequence of base-complementary recognition moieties which can bind specifically to corresponding contiguous bases in the analyte target sequence, and an unbranched, substantially uncharged, substantially stereoregular backbone. A reporter in the system contains (a) a polycationic tail effective to bind electrostatically to the analyte, under conditions in which the reporter does not bind to the substantially uncharged polymers, and (b) reporter groups by which the presence of the reporter can be detected.

Claims

1. A diagnostic system for determination of a single-stranded polynucleotide analyte containing a selected heteromeric target sequence of bases, said system comprising:

a diagnostic reagent composed of a solid support, and attached to the solid support, multiple polymer molecules, each composed of a heteromeric sequence of base-complementary recognition moieties selected from the group consisting of purine and pyrimidine heterocycles adapted to hydrogen-bond to corresponding, contiguous bases in the target sequence, under selected binding conditions, and an unbranched, substantially uncharged backbone, composed of subunit backbone moieties, supporting the recognition moieties at positions which allow hydrogen bonding between the recognition moieties and the corresponding bases in the target sequence, where the subunit backbone moieties are chosen from a group consisting of cyclic moieties, of 5 to 7 atoms in length, and acyclic moieties, of 4 to 6 atoms in length; and

,

molecules of a reporter, where said reporter is composed of an oligocationic tail adapted to bind electrostatically to the charged backbone of the polynucleotide analyte, and attached to the tail, one or more reporter groups adapted to produce a signal by which the presence of the reporter can be detected.

2. The system of claim 1, wherein the polymer molecules have the form:

##STR5## where;

(a) where R₁ -R_n are recognition moieties selected from purine, and pyrimidine heterocycles effective to bind by Watson/Crick pairing to corresponding, contiguous bases in the target sequence, and Bk are backbone moieties;

(b) n is such that the total number of Watson/Crick hydrogen bonds formed between a polymer molecule and target sequence is at least 12;

(c) the backbone moiety length ranges from 5 to 7 atoms if the backbone moieties have a cyclic structure, and 4 to 6 atoms if the backbone moieties have an acyclic structure; and

(d) the backbone moieties support the recognition moieties at positions which allow Watson/Crick base pairing between the recognition moieties and the corresponding, contiguous bases of the target sequences.

3. The system of claim 2, wherein the recognition moieties are selected from the following group:

##STR6## where X=F, CL, Br, or I.

4. The system of claim 2, wherein Bk is a cyclic moiety and Bk≈

Bk are backbone moieties joined predominantly by chemically stable, uncharged, achiral linkages having one of the following forms;

##STR7## where Y=O, S and ##STR8## and R is a selected recognition moiety.

5. The system of claim 2, wherein BK is an acyclic moiety and BK˜

BK are backbone moieties joined predominantly by chemically stable, uncharged, achiral linkages having one of the following forms;

##STR9## where ##STR10## and R is a selected recognition moiety.

6. The system of claim 1, wherein the tail in the reporter contains positive groups at intervals whose spacing corresponds approximately to the spacing between adjacent phosphate groups in the backbone of the polynucleotide analyte.

7. The system of claim 6, wherein the tail contains an oligocationic portion having the formula:

##STR11## where n=2-5, m>

0, and each R₁ -R₅ =H or alkyl group.

8. The system of claim 7, wherein the reporter group in the reporter includes one or more fluorescent, chromophore, enzyme, or radioisotope groups.

11. The system of claim 1, wherein the backbone of the polymer molecule is substantially stereoregular.

9. A method for determination of a polynucleotide analyte containing a selected target base sequence, said method comprising

providing a diagnostic reagent composed of a solid support and, attached to the support, multiple polymer molecules, each composed of a heteromeric sequence of base-complementary recognition moieties selected from the group consisting of purine and pyrimidine heterocycles effective to hydrogen-bond to corresponding, contiguous bases in the target sequence, under selected binding conditions, and an unbranched, substantially uncharged, substantially stereoregular backbone, composed of subunit backbone moieties, supporting the recognition moieties at positions which allow hydrogen bonding between the moieties and the corresponding bases in the target sequence, where the subunit backbone moieties are chosen from the group consisting of cyclic moieties, of 5 to 7 atoms in length, and acyclic moieties, of 4 to 6 atoms in length;

and,

mixing the reagent and an analyte-containing sample under such selected conditions, to produce sequence-specific binding of the analyte to the reagent polymers,

providing a reporter composed of an oligocationic tail adapted to bind electrostatically to the charged backbone of the polynucleotide analyte, and attached to the tail, one or more reporter groups adapted to produce a signal by which the presence of the reporter can be detected,

binding the reporter to analyte which has bound by sequence-specific binding to the reagent, and

determining the extent of reporter binding to the analyte.

10. The method of claim 9, for use in determination of a duplex nucleic acid analyte, wherein said mixing is performed at a selected ionic strength of less than about 0.2M monovalent cation, in the presence of a divalent cation chelating agent and at a temperature above the melting temperature of such duplex analyte.