Nucleic acid multimers and amplified nucleic acid hybridization assays using same

US 5,124,246 A
Filed: 04/18/1989
Issued: 06/23/1992
Est. Priority Date: 10/15/1987
Status: Expired due to Term

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First Claim

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1. A synthetic branched nucleic acid multimer having a minimum of three termini comprising a combination of 5'"'"' and 3'"'"' termini comprising:

(a) at least one first single-stranded oligonucleotide unit that is capable of hybridizing specifically to a first single-stranded nucleic acid sequence of interest; and

(b) a multiplicity of second single-stranded oligonucleotide units that are capable of hybridizing specifically to a second single-stranded nucleic acid sequence of interest, wherein the first single-stranded oligonucleotide unit is bonded directly or indirectly to the multiplicity of second single-stranded oligonucleotide units only via covalent bonds.

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Abstract

Linear or branched oligonucleotide multimers useful as amplifiers in biochemical assays which comprise (1) at least one first single-stranded oligonucleotide unit that is complementary to a single-stranded oligonucleotide sequence of interest, and (2) a multiplicity of second single-stranded oligonucleotide units that are complementary to a single-stranded labeled oligonucleotide. Amplified sandwich nucleic acid hybridizations and immunoassays using the multimers are exemplified.

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59 Claims

1. A synthetic branched nucleic acid multimer having a minimum of three termini comprising a combination of 5'"'"' and 3'"'"' termini comprising:
- (a) at least one first single-stranded oligonucleotide unit that is capable of hybridizing specifically to a first single-stranded nucleic acid sequence of interest; and
  
  (b) a multiplicity of second single-stranded oligonucleotide units that are capable of hybridizing specifically to a second single-stranded nucleic acid sequence of interest, wherein the first single-stranded oligonucleotide unit is bonded directly or indirectly to the multiplicity of second single-stranded oligonucleotide units only via covalent bonds.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 57)
- - 2. The nucleic acid multimer of claim 1 wherein the first oligonucleotide unit has a different sequence than the second oligonucleotide unit and the number of second oligonucleotide units is at least about two-fold the number of first oligonucleotide units.
  - 3. The nucleic acid multimer of claim 1 wherein the first oligonucleotide unit is of about 15 to 50 bases and has a GC content in the range of about 40% to about 60% and the second oligonucleotide unit is of about 15 to 50 bases and has a GC content in the range of about 40% to about 60%.
  - 4. The nucleic acid multimer of claim 1 wherein the nucleotide sequence of the first oligonucleotide unit is the same as the nucleotide sequence of the second oligonucleotide unit and the combined number of units is at least about 3.
  - 5. The nucleic acid multimer of claim 1 wherein the multimer has a "comb-like" branched structure and comprises an oligonucleotide backbone with one or more pendant chains composed of one or more oligonucleotide units.
  - 6. The nucleic acid multimer of claim 1 wherein the multimer has a "fork-like" branched structure.
  - 7. The nucleic acid multimer of claim 1 wherein the multimer is a DNA multimer.
  - 8. The nucleic acid multimer of claim 1 wherein the multimer is an RNA multimer.
  - 9. The nucleic acid multimer of claim 1 wherein either or both of the first and second single-stranded oligonucleotide units contains one or more modified nucleotides.
  - 10. The nucleic acid multimer of claim 1 wherein the oligonucleotide units of the multimer are linked to each other through phosphodiester bonds.
  - 11. The nucleic acid multimer of claim 1 wherein the oligonucleotide units of the multimer are linked to each other via a cross-linking agent.
  - 12. The nucleic acid multimer of claim 11 wherein the cross-linking agent is covalently bonded to the oligonucleotide units of the multimer via N⁴ -modified cytosine bases.
  - 13. The nucleic acid multimer of claim 1 wherein at least a portion of the oligonucleotide units of the multimer are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR10## where R is an organic moiety, R¹ is a hydroxyl protecting group that can be removed under conditions that do not remove synthetic nucleic acid from a solid phase and do not remove exocyclic nitrogen or phosphate protecting groups, X is a phosphorous-containing group that facilitates nucleic acid synthesis, Y is a radical derived from a nucleophilic group, and R² is R¹ or a blocking or protective group that can be removed and replaced with hydrogen without affecting R¹.
  - 14. The nucleic acid multimer of claim 1 wherein at least a portion of the oligonucleotide units are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR11## where Z is a nucleophile, R¹ is a protective group that is generally base-stable and acid sensitive, R² is hydrogen or methyl, R³ is a protective group that can be removed and replaced with hydrogen without affecting R¹, R⁵ is a phosphorus derivative that enables addition of nucleotides to the 5'"'"' position of an oligonucleotide chain during chemical synthesis, R⁶ is methyl, hydrogen, I, Br, or F, and X is an integer in the range of 1 to 8, inclusive.
  - 15. The nucleic acid multimer of claim 1 wherein at least a portion of the oligonucleotide units are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR12## where R is a hydroxyl protecting group, iPr is isopropyl, and R¹ is methyl or beta-cyanoethyl.
  - 16. The nucleic acid multimer of claim 1 wherein the first single-stranded nucleotide sequence of interest is analyte nucleic acid.
  - 17. The nucleic acid multimer of claim 1 wherein the first single-stranded nucleotide sequence of interest is an oligonucleotide that is hybridized to analyte nucleic acid.
  - 18. The nucleic acid multimer of claim 1 wherein the first single-stranded nucleotide sequence of interest is an oligonucleotide that is bound to a ligand.
  - 19. The nucleic acid multimer of claim 1 wherein the second single-stranded nucleotide sequence of interest is a sequence of a single-stranded labeled oligonucleotide.
  - 20. The nucleic acid multimer of claim 1 wherein the second single-stranded nucleotide sequence of interest is a sequence of an oligonucleotide unit of a second nucleic acid multimer.
  - 21. The nucleic acid multimer of claim 1 wherein there is a chemically cleavable linker in each of the second single-stranded oligonucleotide units.
  - 22. The nucleic acid multimer of claim 19 wherein there is a chemically cleavable linker in each of the second single stranded oligonucleotide units.
  - 23. The nucleic acid multimer of claim 21 wherein the linker includes a 1,2-diol group that is cleavable with a periodate.
  - 24. The nucleic acid multimer of claim 22 wherein the linker includes a 1,2-diol group that is cleavable with a periodate.
  - 25. The nucleic acid multimer of claim 23 wherein the linker has the formula:
    - ##STR13##
  - 26. The nucleic acid multimer of claim 24 wherein the linker has the formula:
    - ##STR14##
  - 27. A nucleic acid hybridization assay wherein:
    - (a) the multimer of claim 19 is hybridized via the first oligonucleotide unit to single-stranded analyte nucleic acid bound to a solid phase or to a single-stranded oligonucleotide bound to the analyte, or to a second multimer bound to the analyte;
      
      (b) unbound multimer is removed;
      
      (c) single-stranded labeled oligonucleotide is hybridized to the multimer via the second oligonucleotide(d) unbound labeled oligonucleotide is removed;
      
      (e) the presence of label bound to the multimer is detected.
  - 28. The assay of claim 27 wherein the first oligonucleotide unit has a different sequence than the second oligonucleotide unit and the number of second oligonucleotide units is at least about two-fold the number of first oligonucleotide units.
  - 29. The assay of claim 27 wherein the first oligonucleotide unit is of about 15 to 50 bases and has a GC content in the range of about 40% to about 60% and the second oligonucleotide unit is of about 15 to 50 bases and has a GC content in the range of about 40% to about 60%.
  - 30. The assay of claim 27 wherein the nucleotide sequence of the first oligonucleotide unit is the same as the nucleotide sequence of the second oligonucleotide unit and the combined number of units is at least about 3.
  - 31. The assay of claim 27 wherein the oligonucleotide units of the multimer are linked to each other through phosphodiester bonds.
  - 32. The assay of claim 27 wherein the oligonucleotide units of the multimer are linked to each other via a cross-linking agent.
  - 33. The assay of claim 32 wherein the cross-linking agent is covalently bonded to the oligonucleotide units of the multimer via N⁴ -modified cytosine bases.
  - 34. The assay of claim 32 wherein at least a portion of the oligonucleotide units of the multimer are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR15## where R is an organic moiety, R¹ is a hydroxyl protecting group that can be removed under conditions that do not remove synthetic nucleic acid from a solid phase and do not remove exocyclic nitrogen or phosphate protecting groups, X is a phosphorous-containing group that facilitates nucleic acid synthesis, Y is a radical derived from a nucleophilic group, and R² is R¹ or a blocking or protective group that can be removed and replaced with hydrogen without affecting R¹.
  - 35. The assay of claim 27 wherein at least a portion of the oligonucleotide units are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR16## where Z is a nucleophile, R¹ is a protective group that is generally base-stable and acid sensitive, R² is hydrogen or methyl, R³ is a protective group that can be removed and replaced with hydrogen without affecting R¹, R⁵ is a phosphorus derivative that enables addition of nucleotides to the 5'"'"' position of an oligonucleotide chain during chemical synthesis, R⁶ is methyl, hydrogen, I, Br, or F, and X is an integer in the range of 1 to 8, inclusive.
  - 36. The assay of claim 27 wherein at least a portion of the oligonucleotide units are linked via a multifunctional moiety derived from a compound of the formula:
    - ##STR17## where R is a hydroxyl protecting group, iPr is isopropyl, and R¹ is methyl or beta-cyanoethyl.
  - 38. An immunoassay for an analyte molecule comprising:
    - (a) binding a ligand specifically and directly or indirectly to the analyte, said ligand having a single-stranded oligonucleotide that is complementary to the first oligonucleotide unit of the multimer of claim 1 bound thereto;
      
      (b) removing unbound ligand;
      
      (c) hybridizing the multimer of claim 1 to the ligand;
      
      (d) hybridizing a labeled oligonucleotide to the second oligonucleotide units of the bound multimer;
      
      (e) removing unbound labeled oligonucleotide;
      
      (f) detecting the presence of label bound to the bound multimer.
  - 57. The assay of claim 38 wherein the analyte molecule is an antigen and the ligand is an antibody.

37. A solution sandwich nucleic acid hybridization assay for detecting a first nucleic acid sequence that is part of a nucleic acid segment that includes a second nucleic acid sequence in a sample that contains said segment and another nucleic acid segment that comprises the first nucleic acid sequence but does not include the second nucleic acid sequence comprising:
- (a) contacting the analyte under hybridizing conditions with an excess of (i) an amplifier probe oligonucleotide comprising a first segment that is complementary to one of the first nucleic acid sequence or the second nucleic acid sequence and a second segment that is complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a capture probe oligonucleotide comprising a first segment that is complementary to the other of the first nucleic acid sequence or the second nucleic acid sequence and a second segment that is complementary to an oligonucleotide bound to a solid phase;
  
  (b) contacting under hybridizing conditions the product of step (a) with the oligonucleotide bound to the solid phase;
  
  (c) thereafter separating materials not bound to the solid phase;
  
  (d) contacting under hybridizing conditions the solid phase complex product of step (c) with the nucleic acid multimer, said multimer comprising (i) at least one oligonucleotide unit that is complementary to the second segment of the amplifier probe oligonucleotide and (ii) a multiplicity of second oligonucleotide units that are complementary to a labeled oligonucleotide;
  
  (e) removing unbound multimer;
  
  (f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
  
  (g) removing unbound labeled oligonucleotide; and
  
  (h) detecting the presence of label in the solid phase complex product of step (g).

39. A synthetic linear nonhomopolymeric nucleic acid multimer useful as a means for amplifying a detectable signal in an assay involving nucleic acid hybridization consisting essentially of:
- (a) at least one first single-stranded oligonucleotide unit that is capable of hybridizing specifically to a first single-stranded nucleic acid sequence of interest; and
  
  (b) a multiplicity of second single-stranded oligonucleotide units all of which are capable of hybridizing specifically to a second single-stranded nucleic acid sequence of interest, wherein the first single-stranded oligonucleotide unit is bonded directly or indirectly to the multiplicity of second single-stranded oligonucleotide units only via covalent bonds.
- View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59)
- - 40. The nucleic acid multimer of claim 39 wherein the first oligonucleotide unit has a different sequence than the second oligonucleotide unit and the number of second oligonucleotides units is at least about two-fold the number of first oligonucleotide units.
  - 41. The nucleic acid multimer of claim 39 wherein the first oligonucleotide unit has about 15 to 30 bases and has a GC content in the range of about 40% to about 60% and the second oligonucleotide unit is of about 15 to 30 bases and has a GC content in the range of about 40% to about 60%.
  - 42. The nucleic acid multimer of claim 39 wherein the nucleotide unit sequence of the first oligonucleotide unit is the same as the nucleotide sequence of the second oligonucleotide unit and the combined number of units is at least about 3.
  - 43. The nucleic acid multimer of claim 39 wherein the multimer is a DNA multimer.
  - 44. The nucleic acid multimer of claim 39 wherein the multimer is an RNA multimer.
  - 45. The nucleic acid multimer of claim 39 wherein either or both of the first and second single-stranded oligonucleotide units contains one or more modified nucleotides.
  - 46. The nucleic acid multimer of claim 39 wherein the oligonucleotide units of the multimer are linked to each other through phosphodiester bonds.
  - 47. The nucleic acid multimer of claim 39 wherein the oligonucleotide units of the multimer are linked to each other via a cross-linking agent.
  - 48. The nucleic acid multimer of claim 39 wherein the first single-stranded nucleotide sequence of interest is analyte nucleic acid.
  - 49. The nucleic acid multimer of claim 39 wherein the first single-stranded nucleotide sequence of interest is an oligonucleotide that is hybridized to analyte nucleic acid.
  - 50. The nucleic acid multimer of claim 39 wherein the first single-stranded nucleotide sequence of interest is an oligonucleotide that is bound to a ligand.
  - 51. The nucleic acid multimer of claim 39 wherein the second single-stranded nucleotide sequence of interest is a sequence of a single-stranded labeled oligonucleotide.
  - 52. The nucleic acid multimer of claim 39 wherein the second single-stranded nucleotide sequence of interest is a sequence of an oligonucleotide unit of a second nucleic acid multimer.
  - 53. A nucleic acid hybridization assay wherein:
    - (a) the multimer of claim 51 is hybridized via the first oligonucleotide unit to single-stranded analyte nucleic acid bound to a solid phase or to a single-stranded oligonucleotide bound to the analyte;
      
      (b) unbound multimer is removed;
      
      (c) single-stranded labeled oligonucleotide is hybridized to the multimer via the second oligonucleotide units;
      
      (d) unbound labeled oligonucleotide is remove; and
      
      (e) the presence of label bound to the multimer is detected.
  - 54. The assay of claim 53 wherein the first oligonucleotide unit has a different sequence than the second oligonucleotide unit and the number of second oligonucleotide units is at least about two-fold the number of first oligonucleotide units.
  - 55. The assay of claim 53 wherein the oligonucleotide units of the multimer are linked directly to each other through phosphodiester bonds.
  - 56. The assay of claim 53 wherein the oligonucleotide units of the multimer are linked to each other via a cross-linking agent.
  - 58. An assay for an analyte molecule comprising:
    - (a) binding a ligand specifically and directly or indirectly to the analyte, said ligand having a single-stranded oligonucleotide that is complementary to the first oligonucleotide unit of the multimer of claim 39 bound thereto;
      
      (b) removing unbound ligand;
      
      (c) hybridizing the multimer of claim 39 to the ligand;
      
      (d) hybridizing a labeled oligonucleotide to the second oligonucleotide units of the bound multimer;
      
      (e) removing unbound labeled oligonucleotide;
      
      (f) detecting the present of label bound to the bound multimer.
  - 59. The assay of claim 58 wherein the analyte molecule is an antigen and the ligand is an antibody.

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Current Assignee
Siemens Healthcare Diagnostics Incorporated (Siemens AG)
Original Assignee
Chiron Corporation (Bayer AG)
Inventors
Urdea, Michael S., Horn, Thomas, Warner, Brian
Primary Examiner(s)
Wax, Robert A.
Assistant Examiner(s)
Marschel, Ardin H.

Application Number

US07/340,031
Time in Patent Office

1,162 Days
Field of Search

435/6, 435/810, 436/501, 536/27, 935/23, 935/78, 935/88
US Class Current

435/6.11
CPC Class Codes

C07H 19/06   Pyrimidine radicals

C07H 21/00   Compounds containing two or...

C12Q 1/682   Signal amplification

C12Q 1/689   for bacteria

C12Q 1/706   for hepatitis

G01N 2333/045   Cytomegalovirus

G01N 33/532   Production of labelled immu...

Y10S 435/81   Packaged device or kit

Nucleic acid multimers and amplified nucleic acid hybridization assays using same

First Claim

4 Assignments

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833 Citations

59 Claims

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Nucleic acid multimers and amplified nucleic acid hybridization assays using same

First Claim

4 Assignments

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Abstract

833 Citations

59 Claims

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