Fluorescent N-nucleosides and fluorescent structural analogs of N-nucleosides

• US 6,268,132 B1
• Filed: 03/13/1998
• Issued: 07/31/2001
• Est. Priority Date: 02/12/1992
• Status: Expired due to Fees

• Current Assignee: Chromagen, Inc. (Merloni Holding SPA)
• Original Assignee: Chromagen, Inc. (Merloni Holding SPA)
• Inventors: Conrad, Michael J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/039,021
• Time in Patent Office: 1,236 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/24.5, 536/25.3, 536/25.31, 536/25.32
• US Class Current: 435/6.16
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Structural analogs of the six non-fluorescentN-nucleosides commonly found in RNA and DNA, which are inherently fluorescent under physiological conditions, are identified and methods for their preparation provided. Such analogs may be incorporated into DNA and/or RNA oligonucleotides via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides having prescribed sequences. Such analogous sequences may be identical to, or the analogous complement of, template or target DNA or RNA sequences to which the fluorescent oligonucleotides can be hybridized. Methods of preparing either RNA or DNA oligonucleotide probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in oligonucleotide amplification, detection, identification, and/or hybridization assays are also provided.

Claims

1. An improvement in methods of synthesis, amplification, enzymatic digestion, base-pairing, labeling, sequencing, replication, transcription, location, detection, or identification of DNA or RNA oligonucleotides, wherein said improvement comprises the incorporation of inherently fluorescent nucleosides, or structural analogs thereof, said inherently fluorescent nucleosides or structural analogs thereof being fluorescent under physiological conditions, having the following structures:

wherein

X₁, X₂, X₃, X₄, X₅, and X₆═

N, O, C, S, or Si, wherein at least one of X₁, X₂, X₃, X₄, X₅, or X₆═

N, and wherein X₇ is —

CH—

;

R₄ is a reactive group derivatizable with a detectable label wherein said reactive group is selected from the group consisting of NH₂, SH, ═

O, and optionally, a linking moiety selected from the group consisting of an amide, a thioether, a disulfide, a combination of an amide a thioether or a disulfide, R₁—

(CH₂)_x—

R₂ and R₁—

R₂—

(CH₂)_x—

R₃ wherein x is an integer from 1 to 25 inclusive, and R₁, R₂, and R₃ are H, OH, alkyl, acyl, amide, thioether, or disulfide, and wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent or chemiluminescent reporter molecules, antibodies, haptens, biotin, photobiotin, digoxigenin, fluorescent aliphatic amino groups, avidin, enzymes, and acridinium;

R₅ is H, absent, or part of an etheno linkage with R₄;

R₆ is H, NH₂, SH, or ═

O;

R₈ and R₉ are hydrogen, methyl, bromine, fluorine, or iodine;

alkyl or aromatic substituents, or an optional linking moiety selected from the group consisting of an amide, a thioether, a disulfide linkage, and a combination thereof;

R₁₀ is hydrogen, an acid-sensitive/base-stable blocking group, or a phosphorous derivative;

R₁₁═

R₁₃═

H;

R₁₂ is hydrogen, OH, 3 amino, 3-azido, 3-thiol, 3-unsaturated or a 3-phosphorous derivative; and

R₁₄ is H, OH, or OR₃ where R₃ is a reactive group, protecting group, or additional fluorophore;

provided that excluded from such structure is any purine-like compound in which;

(i) X₁═

X₄═

C;

X₂═

X₃═

N;

R₄═

NH₂;

R₅═

R₈ which is absent;

R₆═

H;

R₉ is H or is absent;

R₁₀═

H; and

R₁₂═

R₁₄═

OH;

or

(ii) X₁═

C;

X₂═

X₃═

X₄═

N;

R₄═

NH₂ or H;

R₅═

R₈ which is absent;

R₆═

NH₂;

R₉ is H or is absent;

R₁₀═

H; and

R₁₂═

R₁₄OH;

or

(iii) R₄ and R₅ in combination form an etheno linkage;

R₆═

R₈═

H;

R₉ is absent;

X₁═

X₃═

C; and

X₂═

X₄═

N;

or

(iv) X₁═

X₂═

C;

X₃═

X₄═

N;

R₄═

halogen or —

S(CH₂)_nR with n being an integer between 1-6 and R is lower alkoxy, alkylthio, phenoxy, phenylthio, unsubstituted or substituted phenyl, —

C═

C—

R′

wherein R′

is unsubstituted or mono-, di- or trisubstituted phenyl;

R₉═

R₁₀═

H; and

R₁₂═

R₁₄═

acyloxy;

or

(v) R₄═

NH₂ or OH;

R₅ is absent;

R₉ is —

COOH, —

CONH₂, —

C(S)NH₂, —

C(NH)N₂, or —

C(N—

NH₂)NH₂;

X₁═

X₂═

X₃═

C; and

X₄═

N;

and further provided that R₈ is absent if X₃═

N and R₉ is absent if X₂═

N;

and provided that the structure is not a nucleoside which is effectively non-fluorescent under physiological conditions and is selected from the group consisting of adenosine, cytidine, guanine, thymidine, uridine, and inosine.

2. The methods of claim 1, wherein said synthesis, amplification, labeling, detection, or identification of DNA or RNA oligonucleotides is by (i) chemical synthesis, polymerization, or linking methods;

or (ii) enzymatic methods of amplification replication, endonuclease DNA or RNA digestion, transcription, terminal labeling, filling in, or nick translation.