Reagents for detecting and assaying nucleic acid sequences

• US 5,175,270 A
• Filed: 09/10/1986
• Issued: 12/29/1992
• Est. Priority Date: 09/10/1986
• Status: Expired due to Term

• Current Assignee: CF Partners Acquisition III, LLC
• Original Assignee: Polyprobe, Inc.
• Inventors: Nilsen, Thor W., Prensky, Wolf
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US06/906,222
• Time in Patent Office: 2,302 Days
• Field of Search: 435/6, 435/91, 435/291, 436/501, 436/811, 536/27-29, 935/32, 935/78
• US Class Current: 536/24.32
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

A novel class of reagents for assaying nucleic acid sequences comprise successive layers of polynucleotides of a specific structure, including a double-stranded waist and single-stranded, free arms at the molecule ends, formed by hybridization of the arms to adjacent molecule arms. The reagent is used to assay for specific nucleic acid sequences. The outer layer of polynucleotides are specific for the sequence to be assayed through their non-annealed, free, single-stranded arms. The reagents are useful in the assay of a wide variety of nucleic acid sequences including those associated with pathogens such as pathogenic bacteria and virus.

Claims

1. A dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein each matrix polynucleotide monomer, prior to hybridization bonding, has a linear, double stranded waist region having a first end and a second end, said first end terminating with two single stranded hybridization regions, each from a strand of the waist region, and said second end terminating with one or two single stranded hybridization region(s), each from a strand of the waist region; and

, the plurality of matrix polynucleotide monomers present does not exceed saturation of the dendritic polynucleotide.

2. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising the dendritic polynucleotide of claim 1 wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

9. A composition according to claim 2, wherein the single stranded hybridization arms each contain from 12 to 1000 nucleotides.

10. A composition according to claim 2, wherein the single stranded hybridization arms each contain from 20 to 100 nucleotides.

11. A composition according to claim 2, further comprising a label.

12. A composition according to claim 2, wherein the plurality of single stranded hybridization arms comprise at least 12 single stranded hybridization arms.

14. A composition according to claim 2, wherein said plurality of the single stranded hybridization arms includes at least one single stranded hybridization arm complementary to at least one single stranded hybridization arm of a second composition comprising a second dendritic polynucleotide having a plurality of single stranded hybridization arms;

said second dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said second dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded; and

, wherein in said second dendritic polynucleotide, each matrix polynucleotide monomer, prior to hybridization bonding, has a linear, double stranded waist region having a first end and a second end, said first end terminating with two single stranded hybridization regions, each from a strand of the waist region, and said second end terminating with one or two single stranded hybridization region(s), each from a strand of the waist region; and

the plurality of matrix polynucleotide monomers present does not exceed saturation of the second dendritic polynucleotide.

15. A composition according to claim 2, wherein said plurality of the single stranded hybridization regions are complementary to a nucleic acid sequence of a pathogen.

16. A composition according to claim 15, wherein the pathogen is a HIV-I virus.

17. A composition according to claim 2, wherein the polynucleotides contain deoxyribonucleic acid (DNA).

18. A composition according to claim 2, wherein the polynucleotides contain ribonucleic acid (RNA).

19. A composition according to claim 2, wherein the polynucleotides contain both RNA and DNA.

20. A composition according to claim 2, wherein two bonded matrix polynucleotide monomers are crosslinked to each other.

21. A composition according to claim 20, wherein the two matrix polynucleotide monomers are crosslinked by exposure to ultraviolet light.

22. A composition according to claim 2, having approximately 100,000-200,000 bases.

23. A composition according to claim 2 having approximately 1×

10⁶ to 1×

10⁷ bases.

24. A composition according to claim 2, having a sphere volume of at least 4.1×

10⁵ (nm)³ and a total DNA concentration of at least 1 mg/ml.

25. A composition according to claim 2 additionally comprising a non-nucleic acid support bound to the composition.

26. A composition according to claim 25, wherein said plurality of the single stranded hybridization arms are complementary to two or more nucleic acid sequences.

27. A composition according to claim 25, wherein said plurality of the single stranded hybridization arms are complementary to a nucleic acid sequence of a pathogen.

28. A composition according to claim 27, wherein t he pathogen is a HIV-I virus.

29. A composition according to claim 14 additionally comprising a non-nucleic acid support bound to the first or second composition.

3. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to be hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected; and

, wherein the plurality of single stranded hybridization arms comprises from at least 108 single stranded arms, up to and including, but not exceeding, that which saturates the dendritic polynucleotide.

13. A composition according to claim 3, wherein the plurality of single stranded hybridization arms comprise at least 972 single stranded hybridization arms.

4. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein said composition has a sphere volume in a range of 1.6×

10⁶ (nm)³ to 7.5×

10⁸ (nm)³, but the sphere volume does not exceed saturation; and

wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

5. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein the composition has a shell volume in a range of 1.2×

10⁶ (nm)³ to 1.0×

10⁸ (nm)³, but does not exceed saturation; and

, wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

6. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprises of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein said dendritic polynucleotide comprises DNA and the composition has a total DNA volume in a range of 5.0×

10² (nm)³ to 2.0×

10¹¹ (nm)³, but does not exceed saturation; and

, wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

7. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprises of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein said dendritic polynucleotide comprises DNA and the composition has a total DNA concentration in a range of 1 mg/ml to 744 mg/ml, but does not exceed saturation; and

, wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

8. A composition for use as a hybridization reagent for detection of a nucleic acid sequence, said composition comprising a dendritic polynucleotide having a plurality of single stranded hybridization arms;

said dendritic polynucleotide being comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein said composition has a shell concentration in a range of 1 mg/ml to 899 mg/ml, but does not exceed saturation; and

, wherein the plurality of single stranded hybridization arms includes at least one sequence complementary to the nucleic acid sequence to be detected.

30. A saturated composition comprising a dendritic polynucleotide comprised of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being hybridization bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in said dendritic polynucleotide each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein the number of non-bonded single stranded hybridization arms multiplied by the volume of one matrix polynucleotide monomer is greater than the space available for bonding of the matrix polynucleotide monomers to the non-bonded single stranded hybridization arms; and

, wherein each matrix polynucleotide monomer, prior to hybridization bonding, has a linear double stranded waist region having a first end and a second end, said first end terminating with two single stranded hybridization regions, each from a strand of the waist region, and said second end terminating with one or two single stranded hybridization region(s), each from a strand of the waist region; and

the plurality of matrix polynucleotide monomers present does not exceed saturation of the dendritic polynucleotide.

31. A composition according to claim 30, wherein the single stranded hybridization arms each contain from 12 to 1000 nucleotides.

32. A composition according to claim 30, wherein the single stranded hybridization arms each contain from 20 to 100 nucleotides.

33. A composition according to claim 30, further comprising a label.

34. A composition according to claim 30, wherein the non-bonded single stranded hybridization arms comprise at least 972 single stranded hybridization arms.

35. A composition according to claim 30, wherein said plurality of the single stranded hybridization arms of the saturated composition includes at least one single stranded hybridization arm complementary to at least one single stranded hybridization arm of a second composition comprising a plurality of dendritic polynucleotides, each dendritic polynucleotide of said second composition being comprises of a plurality of matrix polynucleotide monomers bonded together by hybridization;

each matrix polynucleotide monomer, prior to being bonded to other matrix polynucleotide monomers, having at least three single stranded hybridization regions; and

in each dendritic polynucleotide of said second composition each matrix polynucleotide monomer is hybridization bonded to at least one other matrix polynucleotide monomer at at least one such hybridization region and when hybridization bonded to more than one such hybridization region of the same matrix polynucleotide monomer, there is an intermediate region where the two monomers are not hybridization bonded;

wherein prior to forming the second composition each dendritic polynucleotide thereof has at least three single stranded hybridization arms, and in the second composition each dendritic polynucleotide is hybridization bonded to at least one other dendritic polynucleotide at at least one such hybridization arm, and when bonded to more than one such hybridization arm of the same dendritic polynucleotide there is an intermediate region where the two dendritic polynucleotides are not bonded, at least one single stranded hybridization arm of said second composition is not bonded to any other dendritic polynucleotide of said second composition, and at least one such non-bonded single stranded hybridization arm of said second composition is complementary to the at least one single stranded hybridization arm of the saturated composition; and

, wherein in said second composition, each matrix polynucleotide monomer, prior to hybridization bonding, has a linear, double stranded waist region having a first end and a second end, said first end terminating with two single stranded hybridization regions, each from a strand of the waist region, and said second end terminating with one or two single stranded region(s), each from a strand of the waist region; and

, the plurality of matrix polynucleotide monomers present does not exceed saturation of the second composition.

36. A composition according to claim 30, wherein said plurality of the single stranded hybridization arms are complementary to a nucleic acid sequence to be detected.

37. A composition according to claim 30, wherein said plurality of the single stranded hybridization arms are complementary to a nucleic acid sequence of a pathogen.

38. A composition according to claim 37, wherein the pathogen is a HIV-I virus.

39. A composition according to claim 30, wherein the dendritic polynucleotides contain deoxyribonucleic acid (DNA).

40. A composition according to claim 30, wherein the dendritic polynucleotides contain ribonucleic acid (RNA).

41. A composition according to claim 30, wherein the dendritic polynucleotides contain both RNA and DNA.

42. A composition according to claim 30, wherein two bonded dendritic matrix polynucleotide monomers are crosslinked to each other.

43. A composition according to claim 42, wherein the two dendritic matrix polynucleotide monomers are crosslinked by exposure to ultraviolet light.

44. A composition according to claim 30, having approximately 100,000-200,000 bases.

45. A composition according to claim 30, having approximately 1×

10⁶ to 1×

10⁷ bases.

46. A composition according to claim 30, having a sphere volume in a range of 1.6×

10⁶ (nm)³ to 7.5×

10⁸ (nm)³, but the sphere volume does not exceed saturation.

47. A composition according to claim 30, having a shell volume in a range of 1.2×

10⁶ (nm)³ to 1.0×

10⁸ (nm)³, but does not exceed saturation.

48. A composition according to claim 30, wherein the dendritic polynucleotide comprises DNA and the composition has a total DNA volume in a range of 5.0×

10² (nm)³ to 2.0×

10¹¹ (nm)³, but does not exceed saturation.

49. A composition according to claim 30, wherein the dendritic polynucleotide comprises DNA and the composition has a total DNA concentration in a range of 1 mg/ml to 744 mg/ml, but does not exceed saturation.

50. A composition according to claim 30, having a shell concentration in a range of 1 mg/ml to 899 mg/ml, but does not exceed saturation.

51. A composition according to claim 30, wherein the dendritic polynucleotide comprises DNA and the composition has a sphere volume of at least 4.1×

10⁵ (nm)³ and total DNA concentration of at least 1 mg/ml, but saturation is not exceeded.