Methods of making single-stranded circular oligonucleotides

• US 5,426,180 A
• Filed: 01/11/1993
• Issued: 06/20/1995
• Est. Priority Date: 03/27/1991
• Status: Expired due to Fees

• Current Assignee: University of Rochester
• Original Assignee: Research Corporation Technologies
• Inventors: Kool, Eric T.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/004,800
• Time in Patent Office: 890 Days
• Field of Search: 536/25.3, 536/24.3, 536/24.31, 536/24.32, 536/24.5
• US Class Current: 536/25.3
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/152 on a single-stranded target, e.g. fold-back TFOs
• CPC Class Codes: C12N 2310/3183 Diol linkers, e.g. glycols or propanediols
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS

Abstract

The present invention provides single-stranded circular oligonucleotides each with at least one parallel binding (P) domain and at least one corresponding anti-parallel binding (AP) domain separated from each other by loop domains. When more than one P or AP domain is included in a circular oligonucleotide of the present invention, the additional P or AP domains can constitute loop domains for a pair of corresponding P and AP domains, and vice versa. Each P and AP domain has sufficient complementarity to bind to one strand of a defined nucleic acid target wherein the P domain binds in a parallel manner to the target and the corresponding AP domain binds in an anti-parallel manner to the target. Moreover, the present single-stranded circular oligonucleotides can bind to both single-stranded and double-stranded target nucleic acids. The present invention also provides methods of making and using these oligonucleotides as well as kits and pharmaceutical compositions containing these oligonucleotides.

Claims

1. A method of preparing a single-stranded circular oligonucleotide comprising binding a linear precircle to an end-joining-oligonucleotide, joining two ends of said precircle and recovering said single-stranded circular oligonucleotide;

wherein said single-stranded circular oligonucleotide comprises at least one parallel binding (P) domain and at least one anti-parallel binding (AP) domain having a loop domain between each binding domain to form said circular oligonucleotide;

each P and corresponding AP domain having sufficient complementarity to detectably bind to one strand of a defined nucleic acid target wherein said P domain binds in a parallel manner to said target, and said corresponding AP domain binds in an anti-parallel manner to said target.

2. The method of claim 1 wherein said linear precircle has a 3'"'"'-phosphate and a 5'"'"'-OH.

3. The method of claim 2 wherein said two ends are AP nucleotides of said single-stranded circular oligonucleotide.

4. The method of claim 3 wherein said joining is performed with BrCN, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or N-cyanoimidazole ZnCl₂.

5. The method of claim 1 wherein said target comprises a known nucleotide sequence from which a nucleotide sequence for a sufficient number of positions in said P domain and a sufficient number of positions in said AP domain is determined, and wherein, for said P domain:

when a base for a position in said target is guanine or a guanine analog, then P has cytosine, or a suitable analog thereof, in a corresponding position;

when a base for a position in said target is adenine, or an adenine analog then P has thymine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is thymine, or a thymine analog, then P has cytosine or guanine, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is cytosine, or a cytosine analog, then P has cytosine, thymine or uracil, or suitable analogs thereof, in a corresponding position; and

when a base for a position in said target is uracil, or a uracil analog, then P has cytosine, guanine, thymine or uracil, or suitable analogs thereof, in a corresponding position;

and for said AP domain;

when a base for a position in said target is guanine, or a guanine analog, then AP has cytosine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is adenine, or an adenine analog, then AP has thymine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is thymine, or a thymine analog, then AP has adenine, or a suitable analog thereof, in a corresponding position;

when a base for a position in said target is cytosine, or a cytosine analog, then AP has a guanine, or a suitable analog thereof, in corresponding position; and

when a base for a position in said target is uracil, or a uracil analog, then AP has adenine or guanine, or suitable analogs thereof, in a corresponding position; and

wherein said sufficient number of positions provide complementarity for said oligonucleotide to detectably bind to said target.

6. The method of claim 1 wherein said P domain comprises a nucleotide sequence which is determined from a known nucleotide sequence of said target:

when a base for a position in said target is guanine or a guanine analog, then P has cytosine, or a suitable analog thereof, in a corresponding position;

when a base for a position in said target is adenine, or an adenine analog then P has thymine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is thymine, or a thymine analog, then P has cytosine or guanine, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is cytosine, or a cytosine analog, then P has cytosine, thymine or uracil, or suitable analogs thereof, in a corresponding position; and

when a base for a position in said target is uracil, or a uracil analog, then P has cytosine, guanine, thymine or uracil, or suitable analogs thereof, in a corresponding position;

and wherein said AP domain comprises a nucleotide sequence which is determined from said sequence of said target as follows;

when a base for a position in said target is guanine, or a guanine analog, then AP has cytosine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is adenine, or an adenine analog, then AP has thymine or uracil, or suitable analogs thereof, in a corresponding position;

when a base for a position in said target is thymine, or a thymine analog, then AP has adenine, or a suitable analog thereof, in a corresponding position;

when a base for a position in said target is cytosine, or a cytosine analog, then AP has a guanine, or a suitable analog thereof, in corresponding position; and

when a base for a position in said target is uracil, or a uracil analog, then AP has adenine or guanine, or suitable analogs thereof, in a corresponding position.