Probes comprising fluorescent nucleosides and uses thereof

• US 5,652,099 A
• Filed: 08/18/1994
• Issued: 07/29/1997
• Est. Priority Date: 02/12/1992
• Status: Expired due to Fees

• Current Assignee: Michael J. Conrad
• Original Assignee: Michael J. Conrad
• Inventors: Conrad, Michael J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/292,892
• Time in Patent Office: 1,076 Days
• Field of Search: 435/6, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/26.23, 536/26.26, 536/26.6, 536/26.7, 536/26.74, 536/27.13, 536/27.14, 536/27.21, 536/27.6 , 536/28.2, 536/27.7, 536/27.8, 536/27.81, 536/28.5, 536/28.53, 536/28.54, 536/28.55, 935/78
• 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 amplication, detection, identification, and/or hybridization assays are also provided.

Claims

1. A fluorescent nucleoside, or structural analog thereof, having either of the following structures:

##STR2## 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;

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 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 compound 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=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)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)NH2, or --C(N--NH2)NH2;

X1=X2=X3=C; and

X4=N.

2. A polynucleotide probe for the detection or amplification of a target polynucleotide sequence, said probe comprising a fluorescent nucleoside having either of the following structures:

##STR3## 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;

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 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 compound 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=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)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)NH2, or --C(N--NH2)NH2;

X1=X2=X3=C; and

X4=N.

3. A method for detecting a target polynucleotide sequence, said method comprising contacting a sample suspected of having said target sequence with an effective amount of a composition comprising a probe in which, as a substitute for any of the six commonly occurring non-fluorescent N-nucleotides, there is provided a fluorescent nucleotide, under conditions which permit hybridization;

and detecting any hybridization between said probe and said target sequence by observing fluorescence or changes in fluorescence, wherein said fluorescent nucleotide has a structure represented by either of the following structures;

##STR4## 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;

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 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 compound 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=R14=OH;

or

(iii) R4and 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)NH2, or --C(N--NH2)NH2;

X1=X2=X3=C; and

X4=N.

4. The method, according to claim 3, which comprises the simultaneous detection of multiple sites in a genome.