Fluorescent universal nucleic acid end label

• US 5,728,525 A
• Filed: 06/02/1995
• Issued: 03/17/1998
• 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): Horlick, Kenneth R.
• Application Number:
  US08/459,890
• Time in Patent Office: 1,019 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/26.7, 536/26.8, 435/6, 435/91.1
• 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-fluorescent N-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. A 5'"'"' universal end label for a nucleic acid sequence, wherein said label comprises a 5'"'"' homopolymer, said homopolymer comprising a fluorescent nucleoside analog not capable of forming hydrogen-bonded Watson-Crick base pairs, and a sequence complementary to a promoter region on a nucleic acid sequence, wherein said fluorescent nucleoside analog has the structure:

##STR3## wherein X₁ ═

X₆ ═

C or Si;

X₂, X₃, X₄, X₅ ═

C, S, N or Si;

provided that at least one of X₁, X₂, X₃, X₄, X₅, or X₆ ═

N;

X₇ ═

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

R₆ is H, NH₂, SH, or ═

O;

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

an alkyl or aromatic substituent, 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 phosphorus derivative;

R₁₁ ═

R₁₃ ═

H;

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

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

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

(i) X1═

X4═

C;

X2═

X3═

N;

R4═

NH2;

R5═

R8 which is absent;

R6═

H;

R9is H or absent;

R10═

H; and

R12═

R14═

OH;

or

(ii) X1═

C;

X2═

X3═

X4═

N;

R4═

NH2or H;

R5═

R8 which is absent;

R6═

NH2;

R9 is H or is absent;

R10═

H; and

R12═

R14═

OH;

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)_n R, 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 on-, 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)NH2, or --C(N-- NH2)NH2;

X1═

X2═

X3═

C; and

X4═

N.

2. The universal end label, according to claim 1 further having a nucleic acid sequence disposed between and connected to said homopolymer sequence and said sequence complementary to a promoter region.

3. The universal end label, according to claim 1, wherein said universal end label further comprises a nucleic acid sequence complementary to a target sequence which is different from said promoter region.

4. A method for synthesizing a fluorescent 5'"'"' end label probe comprising a 5'"'"' homopolymer, said homopolymer comprising a fluorescent nucleoside analog not capable of forming hydrogen-bonded Watson-Crick base pairs, according to claim 1 and a sequence complementary to a promoter region or a nucleic acid sequence, said method comprising the steps of

(a) restricting, with a specific restriction enzyme, a sequence having a known promoter site and known restriction site downstream from the known promoter site;

(b) inserting a unique target sequence at the restriction site;

(c) hybridizing a fluorescent nucleoside analog probe comprising a sequence complementary to the promoter of the inserted target sequence; and

(d) extending the probe sequence from the hybridized promoter region, using a nucleic acid polymerase, to synthesize a specific probe complementary to the inserted target sequence.

5. A method for increased sensitivity of detection of a target nucleotide sequence, said method comprising

(a) chemically synthesizing a 5'"'"' universal end label according to claim 1;

(b) enzymatically synthesizing a plurality of nucleic acid sequences complementary to a discreet segment of said target sequence, wherein each of said plurality of nucleic acid sequences comprises an end label of step (a);

(c) providing as a cocktail a mixture of the plurality of 5'"'"' end label nucleic acid sequences; and

(d) hybridizing the labeled nucleic acid sequences to a target nucleic acid sequence.

6. A method for determining the sequence of bases in a polynucleotide, said method comprising

(a) synthesizing a plurality of nucleic acid fragments each having different lengths, wherein each of the nucleic acid fragments has attached thereto a 5'"'"' universal end label according to claim 1;

(b) separating on a gel each of the different lengths of said nucleic acid fragments; and

(c) detecting directly on the gel, each of said fragment lengths.