Polynucleotide reagent containing chiral subunits and methods of use

• US 5,521,063 A
• Filed: 02/09/1993
• Issued: 05/28/1996
• Est. Priority Date: 03/15/1985
• Status: Expired due to Term

• Current Assignee: AntiVirals, Inc. (Sarepta Therapeutics, Inc.)
• Original Assignee: AntiVirals, Inc. (Sarepta Therapeutics, Inc.)
• Inventors: Weller, Dwight D., Summerton, James E.
• Primary Examiner(s): BICKEL, MINDY B
• Application Number:
  US08/015,211
• Time in Patent Office: 1,204 Days
• Field of Search: 544/81-2, 544/118, 544/123, 528/398-9, 528/403, 528/405-6, 435/6
• US Class Current: 435/6.12
• CPC Class Codes: A61K 47/545 Heterocyclic compounds A61K47/558 takes precedence
• CPC Class Codes: A61K 47/548 Phosphates or phosphonates, e.g. bone-seeking phospholipids A61K47/544
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• 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: C08G 79/02 a linkage containing phosphorus
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification

Abstract

The present invention describes an assay system wherein target polynucleotide molecules are captured on a support by base-specific binding to support-bound polymers, which are themselves substantially uncharged, and the target polynucleotides can be detected on the basis of their backbone charge. The assay system may also include polycationic reporter molecules which are designed to bind to the fully charged analyte backbone, but not the uncharged (or substantially uncharged) polymer backbone. In one embodiment, the reporter molecules are composed of a polycationic moiety or tail designed to bind electrostatically to a fully charged polynucleotide, under conditions where the reporter does not bind to the less charged or uncharged binding polymer carried on the diagnostic reagent.

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 contain morpholino subunit structures of the form;

##STR2## where (i) the structures are linked together by uncharged, phosphorous-containing chiral linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5'"'"', exocyclic carbon of an adjacent subunit, and (ii) P_i is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide, 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:

##STR3## 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 5-7 atoms; 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 sequence.

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

##STR4## where X is H, CH₃, F, Cl, Br or I.

4. The system of claim 2, wherein the backbone moieties are joined by linkages having at least one of the following forms:

##STR5##

5. The system of claim 4, wherein the linkage is of the form:

##STR6## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and

, R is H, CH₃, or other moiety that does not interfere with said base specific hydrogen bonding.

6. The system of claim 4, wherein the linkage is of the form:

##STR7## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and

, R is H, CH₃, or other moiety that does not interfere with said base specific hydrogen bonding.

7. The system of claim 4, wherein the linkage is of the form:

##STR8## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

Z is O or S.

8. The system of claim 4, wherein the linkage is of the form:

##STR9## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

Z is O or S.

9. The system of claim 4, wherein the linkage is of the form:

##STR10## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and

, R is H, CH₃, or other moiety which does not interfere with said base specific hydrogen bonding.

10. The system of claim 4, wherein the linkage is of the form:

##STR11## where P_j is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; and

,

X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and

, R is H, CH₃, or other moiety which does not interfere with said base specific hydrogen bonding.

11. 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.

12. The system of claim 11, wherein the tail contains an oligocationic portion having the formula:

space="preserve" listing-type="equation">----[N.sup.+ (R.sub.1)(R.sub.2)--(CH.sub.2).sub.n ].sub.m ----N.sup.+ (R.sub.3)(R.sub.4)(R.sub.5)

where n=2-5, m>

0, and each R₁ -R₅ =H or alkyl group.

13. The system of claim 1, wherein the reporter group in the reporter includes one or more fluorescent, chromophore, enzyme, or radioisotope groups.

14. A method for determination of a polynucleotide analyte containing a selected target base sequence using a reporter, 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 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 contain morpholino subunit structures of the form:

##STR12## where (i) the structures are linked together by uncharged, phosphorous-containing chiral linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5'"'"' exocyclic carbon of an adjacent subunit, and (ii) P_i is a purine or pyrimidine base-pairing moiety effective to bind by base-specific hydrogen bonding to a base in a polynucleotide; 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.