Modifications for antisense compounds

• US 10,087,445 B2
• Filed: 11/23/2016
• Issued: 10/02/2018
• Est. Priority Date: 03/26/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Inventors: Behlke, Mark Aaron, Owczarzy, Richard, You, Yong, Walder, Joseph Alan, Lennox, Kim
• Primary Examiner(s): Zara, Jane J
• Application Number:
  US15/360,382
• Publication Number:
  US 20170204412A1
• Time in Patent Office: 678 Days
• Field of Search: 435 61, 435 611, 435 911, 435 9131, 435 612, 536 231, 536 243, 536 245, 536 266, 436501
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/352 linked to the nucleic acid via a carbon atom
• CPC Class Codes: C12N 2310/3529 Aromatic substituent
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

Claims

1. A method of detecting a target oligonucleotide in a sample, the method comprising:

(a) contacting the sample with a composition comprising a probe oligonucleotide capable of hybridizing with the target oligonucleotide, the probe oligonucleotide having the structure 5′

-Y₁—

X—

Y₂-3′

, wherein

Y₁ comprises a fluorophore at or near the 5′

-terminus of the probe oligonucleotide and a sequence of 8-12 DNA or RNA nucleotides comprising a nucleotide N₁ having a 3′

phosphate covalently linked to X;

Y₂ comprises a quencher located at or near the 3′

-terminus of the probe oligonucleotide and a sequence of DNA or RNA nucleotides comprising a nucleotide N₂ having a 5′

phosphate covalently linked to X; and

X comprises an internal quencher;

wherein fluorescence of the fluorophore is reduced when the probe oligonucleotide is not hybridized to the target oligonucleotide; and

(b) detecting the presence of the target oligonucleotide in the sample when an increase in fluorescence of the composition is detected as compared to the fluorescence of the composition in a control sample devoid of the target oligonucleotide.

2. The method of claim 1, wherein the quencher located at or near the 3′

-terminus of the probe oligonucleotide is dabcyl, Eclipse®

quencher, Black Hole quencher BHQ1, Black Hole quencher BHQ2, Black Hole quencher BHQ3, Iowa Black®

FQ, Iowa Black®

RQ-n1, or Iowa Black®

RQ-n2.

3. The method of claim 1, wherein the internal quencher is dabcyl, Eclipse®

quencher, Black Hole quencher BHQ1, Black Hole quencher BHQ2, Black Hole quencher BHQ3, Iowa Black®

FQ, Iowa Black®

RQ-n1, or Iowa Black®

RQ-n2.

4. The method of claim 1, wherein fluorescence of the fluorophore is reduced by fluorescence resonance energy transfer, ground state quenching, or a combination thereof when the probe oligonucleotide is not hybridized to the target oligonucleotide.

5. The method of claim 1, wherein the increase in fluorescence arises from cleavage of the probe oligonucleotide.

6. The method of claim 1, wherein the probe oligonucleotide forms a random-coil conformation when the probe oligonucleotide is unhybridized, such that the fluorescence of the fluorophore is reduced.

7. The method of claim 1, wherein the probe oligonucleotide comprises a self-complementary sequence and wherein the quencher and the fluorophore are attached to the probe oligonucleotide such that the fluorescence of the fluorophore is quenched when the probe oligonucleotide undergoes intramolecular base pairing.

8. The method of claim 1, wherein the method is used in a polymerase chain reaction (PCR), wherein synthesis of PCR product results in an increase in fluorescence.

9. The method of claim 1 wherein the stability of an oligonucleotide duplex comprising the probe oligonucleotide is greater than the stability of an oligonucleotide duplex comprising a comparator probe oligonucleotide lacking an internal quencher.

10. The method of claim 1, wherein Y₁ comprises a fluorophore at the 5′

-terminus of the probe oligonucleotide and a sequence of 9 DNA or RNA nucleotides comprising a nucleotide N₁ having a 3′

phosphate covalently linked to X.