Arrays with modified oligonucleotide and polynucleotide compositions

• US 6,087,112 A
• Filed: 09/29/1999
• Issued: 07/11/2000
• Est. Priority Date: 12/30/1998
• Status: Expired due to Term

• Current Assignee: Lakewood Amedex Inc.
• Original Assignee: Oligos Etc., Inc.
• Inventors: Dale, Roderic M. K.
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): WANG, ANDREW J
• Application Number:
  US09/408,088
• Time in Patent Office: 286 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: C07B 61/00 Other general methods
• 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

Abstract

The present invention provides arrays having associated modified oligonucleotides, e.g., 2'"'"'-O-R oligonucleotides, methods of making such arrays, assays for using such arrays, and kits containing such arrays. In one embodiment, the arrays of the invention exhibit an increased binding affinity with complementary nucleic acids, and in particular with complementary RNA. In another embodiment, the associated nucleic acids of the array of the invention exhibit substantial acid resistance, allowing the arrays to be treated with low pH solutions. In another embodiment, the modified associated nucleic acids of the array of the invention exhibit substantial resistance to nuclease degradation.

Claims

1. An array comprising a plurality of modified oligonucleotide compositions stably associated with the surface of a support, wherein each oligonucleotide composition is characterized by:

an oligonucleotide backbone structure modified from that of a naturally occurring nucleotide polymer;

wherein the oligonucleotides of the composition are characterized by a binding affinity greater than that of a corresponding, non-modified oligonucleotide, wherein the oligonucleotides are further characterized by a pH stability of at least one hour at 37°

C. at a pH range of about 0.5 to about 6.0.

2. The array of claim 1, wherein the oligonucleotides are comprised of a modification at a 2'"'"' site of the sugar group of at least one nucleotide.

3. The array of claim 1, wherein the oligonucleotides further comprise a blocking chemical modification at or near at least one end of said oligonucleotides, and wherein the oligonucleotides are further characterized by a nuclease resistance of at least twice that of a naturally occurring oligonucleotide having the same sequence and number of bases.

4. The array of claim 1, wherein said modified oligonucleotides have an average length of from about 80 to about 300 nucleotides.

5. The array of claim 1, wherein said modified oligonucleotides have an average length of from about 100 to about 200 nucleotides.

6. The array of claim 1, wherein oligonucleotides of each of said oligonucleotide compositions have a different sequence from oligonucleotides of any other oligonucleotide composition on the array.

7. The array of claim 1, wherein each oligonucleotide composition comprises a population of identical oligonucleotides.

8. The array of claim 1, wherein each oligonucleotide composition comprises a plurality of oligonucleotides that bind to a particular nucleic acid.

9. The array of claim 1, wherein the number of oligonucleotide compositions on said array ranges from about 2 to about 10⁹.

10. An array comprising a plurality of modified oligonucleotide compositions stably associated with the surface of a support, wherein each oligonucleotide composition is characterized by:

an oligonucleotide backbone structure modified from that of a naturally occurring nucleotide polymer;

wherein the oligonucleotides of the composition are characterized by a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to about 6.0, and wherein the oligonucleotides are further characterized by having a nuclease resistance of at least twice that of a naturally occurring oligonucleotide having the same sequence and number of bases.

11. The array of claim 10, further comprising a blocking chemical modification at or near at least one end of said oligonucleotide.

12. The array of claim 10, wherein the modified oligonucleotide is further characterized by modification of at least 25% of the internucleoside linkages of the oligonucleotide.

13. An array comprising a plurality of oligonucleotide compositions stably associated with the surface of a support, wherein each oligonucleotide composition is characterized by:

an oligonucleotide backbone structure modified from that of a naturally occurring nucleotide polymer; and

a blocking chemical modification at or near at least one end of the oligonucleotide;

wherein the oligonucleotide is characterized by a nuclease resistance of at least twice that of a naturally occurring polymer having the same number of nucleotides and a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to 6.0.

14. An array of modified oligonucleotides, the array comprising:

a planar, non-porous solid support having a surface;

a plurality of different modified oligonucleotides attached to the surface of the solid support at a density exceeding 400 different modified oligonucleotides/cm²,

wherein each of the different modified oligonucleotides is attached to the surface of the solid support in a different predefined region, has a different determinable sequence, and is at least 80 nucleotides in length; and

further wherein the modified oligonucleotides are characterized by a characteristic selected from the group consisting of (a) a binding affinity of at least about 1.25 times that of a corresponding, non-modified oligonucleotide, (b) a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to 6.0; and

(c) a nuclease resistance of at least twice that of a naturally occurring oligonucleotide having the same sequence and number of bases.

15. A method of analyzing comprising the steps of:

(a) contacting a first sample of naturally occurring nucleic acid sequences with an array comprised of a solid support having bound to its surface a plurality of modified nucleic acid sequences, said bound sequences characterized by acid resistance and nuclease resistance;

(b) allowing sequence of the sample to hybridize to the modified sequence of the array;

(c) analyzing results of the hybridizing;

(d) removing sequences hybridized to sequences of the array using a removing agent selected from the group consisting of a solution having a pH of less than 6.0 and a nuclease which enzymatically destroys natural nucleic acid sequences; and

(e) repeated (a), (b), (c) and (d) with a second sample of naturally occurring nucleic acid sequences.

17. A method for detecting nucleic acid sequences in two or more collections of nucleic acid molecules, the method comprising:

(a) providing an array of modified polynucleotides bound to a solid surface, each said modified polynucleotide comprising a determinable nucleic acid;

(b) contacting the array of modified polynucleotides with;

(i) a first collection of labeled nucleic acid comprising a sequence substantially complementary to a nucleic acid of said array, and

(ii) at least a second collection of labeled nucleic acid comprising a sequence substantially complementary to a modified polynucleotide of said array;

wherein the first and second labels are distinguishable from each other; and

(c) detecting hybridization of the first and second labeled complementary nucleic acids to nucleic acids of said arrays;

wherein the modified oligonucleotides are characterized by a characteristic selected from the group consisting of (a) a binding affinity of at least about 1.25 times that of a corresponding, non-modified oligonucleotide, (b) a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to 6.0; and

(c) a nuclease resistance of at least twice that of a naturally occurring oligonucleotide having the same sequence and number of bases.

16. The method of claim 17, wherein (a), (b), (c) and (d) are repeated a plurality of times with different samples of naturally occurring nucleic acid sequences.

18. A method of using a label to detect hybridization with modified polynucleotide probes of known sequence, said method comprising:

(a) contacting under hybridization conditions a labeled polynucleotide sequence with a collection of modified polynucleotide probes of known sequences wherein said probes are attached to a substrate at known locations and wherein said polynucleotide probes have a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to 6.0; and

(b) determining the sequences of the probes with hybridize with the labeled polynucleotide, said collection comprising at least 100 different probes per square centimeter of substrate.

19. A method of identifying nucleotide differences between the sequence of a target nucleic acid and the sequence of a reference nucleic acid comprising:

a) providing a substrate having at least 1000 different modified polynucleotide probes of known sequence at known locations, attached at a density of at least 10,000 probes per sequence cm, wherein said polynucleotide probes have a pH stability of at least one hour at 37°

C. at a pH in a range of about 0.5 to 6.0; and

;

b) contacting the target nucleic acid with the modified polynucleotide probes attached to the substrate under conditions for high specificity complementary hybridization;

c) determining which modified polynucleotide probes have hybridized with the target nucleic acid; and

d) using a computer to (i) compare the sequence of the reference nucleic acid with the sequences of the modified polynucleotide probes that have hybridized with the target nucleic acid and (ii) identify the nucleotide differences between the sequence of the target nucleic acid and the sequence of the reference nucleic acid.