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WO1999011820A1 - Compositions et procedes d'identification et de quantification d'oligonucleotides a sequence de deletion dans des preparations d'oligonucleotides de synthese - Google Patents

Compositions et procedes d'identification et de quantification d'oligonucleotides a sequence de deletion dans des preparations d'oligonucleotides de synthese Download PDF

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Publication number
WO1999011820A1
WO1999011820A1 PCT/US1998/018084 US9818084W WO9911820A1 WO 1999011820 A1 WO1999011820 A1 WO 1999011820A1 US 9818084 W US9818084 W US 9818084W WO 9911820 A1 WO9911820 A1 WO 9911820A1
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Prior art keywords
oligonucleotide
oligonucleotides
probe
sensor array
deletion sequence
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PCT/US1998/018084
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English (en)
Inventor
Danhua Chen
G. Susan Srivatsa
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Isis Pharmaceuticals, Inc.
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Publication date
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to AU91278/98A priority Critical patent/AU9127898A/en
Publication of WO1999011820A1 publication Critical patent/WO1999011820A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to compositions and methods for the identification and quantitation of a mixture of various deletion sequence oligonucleotides present in a preparation of a synthetic oligonucleotide of length n via
  • hybridization reactions Unlike chromatographic methods, the hybridization reactions of the invention take place in the absence of electrophoresis or any other flow process and are allowed to proceed to equilibrium.
  • the invention may be used to quantitate the deletion sequence oligonucleotide species present in synthetic preparations of a variety of oligonucleotides, as well as preparations of any molecule which is not technically an oligonucleotide but which has a nucleobase sequence and is capable of hybridizing to a nucleic acid (e.g., peptide nucleic acids) .
  • synthetic oligonucleotide preparation i.e., a set of deletion sequence oligonucleotides that have different nucleobase sequences apparently resulting, in each instance, from the deletion of a single base from an oligonucleotide having a nucleobase sequence of length n.
  • nucleoside monomers are attached to a growing oligomer chain one at a time in a repeated series of chemical reactions such as nucleoside monomer coupling, oxidation, capping and detritylation.
  • the stepwise yield for each nucleoside addition is above 99%. Although impressive, such a yield indicates that some amount (less than 1%) of the preparation of the oligomer chain has failed at each nucleoside monomer addition cycle (Smith, Anal . Chem. , 1988, 60 , 381A) .
  • the final yield of full-length oligonucleotide is not 100% in a synthetic preparation thereof and decreases as n (the number
  • oligonucleotide ranging from ( ⁇ -1)-, ( ⁇ -2)-, etc., to 1-mers
  • ⁇ -mer oligonucleotide product is often only about a few
  • the ( ⁇ -1) portion likely consists of the mixture of
  • deletion or internal deletion sequences depending upon the position of the missing base, i.e., either at the 5 1 or 3 '
  • impurities will usually bind to the same target mRNA as the full length sequence but with a slightly lower affinity. Thus, to some extent, such impurities can be considered as part of the active drug component .
  • the internal single base deletion sequence impurities are not expected to hybridize well to the target mRNA and thus will have either little to no biological activity or undesired biological activity.
  • There are potential side effects for the internal single base deletion sequence impurities including the chance that some of the internal single base deletion sequence impurities would be complementary to a non-target mRNA, leading to an unintended biological response. Therefore, the speciation of the single base deletion sequence impurities, particularly the internal ones, is a parameter of the impurity profile of oligonucleotide drugs (Crooke, Antisense Research and
  • HPLC high pressure liquid chromatography
  • oligonucleotides such as phosphorothioates
  • CGE electrophoresis
  • ES/MS ionization mass spectrometry
  • the electrospray mass spectrometry method has been limited to the identification of the full length oligonucleotide (Bayer et al . , Anal . Chem . , 1994, 66, 3858),
  • the oligonucleotides were tailed with poly-dA (12-20 residues) and annealed to a dT-tailed plasmid.
  • the recombinant plasmid was ligated and used to transform competent bacteria. Clones were randomly selected and the region in the recombinant plasmid containing the inserted oligonucleotide was sequenced.
  • a possible problem to this method is that the plasmid and its host bacteria might be biased towards or against the selection of inserts having a particular sequence; therefore, the results arrived at after cloning may be different from the actual distribution of the ( ⁇ -1) population. The complicated procedure and tedious labor
  • this method is limited to ligatable, clonable oligonucleotides, i . e . , phosphodiester oligonucleotides, and
  • compositions and methods of the invention provide the means to distinguish deletion sequence oligonucleotides having related but different nucleobase sequences and to quantitate the amounts of different species of deletion sequence ("target") oligonucleotides present in a mixture thereof.
  • target deletion sequence
  • Such mixtures include, but are not limited to, solutions containing a set of ( ⁇ -1) -mers with a nucleobase
  • the invention is equally applicable to oligodeoxynucleotides as well as oligonucleotides having synthetic chemical alterations, so long as such alterations do not modify the specificity of the oligonucleotide ' s nucleobase sequence for its reverse complement.
  • the present invention relates to compositions and methods for the identification and quantitation of a mixture of various deletion sequence oligonucleotides present in a preparation of a synthetic oligonucleotide of length ⁇ .
  • deletion oligonucleotides may have a variety of nucleobase sequences related to the base sequence of the full-length oligonucleotide.
  • a solution comprising a mixture of various deletion sequence oligonucleotides that have been detectably labeled is contacted to a composition comprising a series of immobilized probe oligonucleotides. For example, a mixture of ( ⁇ -1) deletion sequence
  • oligonucleotides having differing sequences is hybridized to a composition comprising a variety of probe oligonucleotides, each probe oligonucleotide having a nucleobase sequence that is the precise reverse complement of a given ( ⁇ -1) deletion
  • oligonucleotide of length ⁇ having a defined nucleobase sequence.
  • hybridization reaction is conducted under conditions such that each particular ( ⁇ -1) species is allowed to hybridize (bind) specifically, and with high affinity, to its appropriate reverse complement probe.
  • the hybridization reactions are allowed to proceed for a relatively extended period of time in the absence of flow and under other such conditions as are necessary to allow the hybridization reactions to proceed to equilibrium.
  • Unbound oligonucleotides including, for example, ⁇ -mers
  • U.S. Patent 5,700,637 to Southern is stated to describe an apparatus and method for analyzing a polynucleotide sequence .
  • Published PCT patent application WO 98/31836 describes methods, compositions and algorithms for the detection and quantification of nucleic acid species.
  • the methods and compositions of Drmanac are stated to be useful in techniques such as, for example, sequencing by hybridization and detection of nucleic acids from infectious agents.
  • This invention provides new methods and compositions for the identification and quantitation of deletion sequence oligonucleotides.
  • the invention is capable of distinguishing and quantitating a mixture having from three (3) to about fifty (50) oligonucleotides of the same or similar length, each oligonucleotide having a nucleobase sequence that represents a deletion of one or more bases from the sequence of a parent oligonucleotide.
  • the invention is distinct from, and required solutions to a distinct set of technical problems from those found in the development of, e.g., methods and compositions that can distinguish between two, or at most a few, nucleic acids having single base mismatches relative to each other (see, e.g., Wallace et al . , Nucleic Acids Research,
  • the invention may be used to quantitate the deletion sequence oligonucleotide species present in synthetic preparations of a variety of oligonucleotides, as well as preparations of any molecule which is not technically an oligonucleotide but which has a nucleobase sequence and is capable of hybridizing to a nucleic acid (e.g., peptide nucleic acids) .
  • the methods and compositions of the invention are used to characterize the types, and quantitate the amounts, of different ( ⁇ -1) -mers
  • oligonucleotides present in a sample from a preparation of synthetic full length oligonucleotides ( ⁇ -mers) .
  • step (a) comprises isolating a representative
  • Step (b) comprises contacting a mixture
  • oligonucleotides with a composition (which may be a matrix) comprising from 2 to about 50 sensor arrays of the invention.
  • a composition which may be a matrix
  • Optional step (c) comprises washing the matrix
  • Step (d) comprises detecting and
  • compositions of the invention comprise a series of sensor arrays, each of which comprises a "probe" oligonucleotide having a unique nucleobase sequence.
  • Each sensor array preferably comprises up to four parts.
  • Part (1) provides a solid support for the other parts (e.g., a glass slide).
  • Part (2) is a first linker (e.g., a hexylamino group)
  • Part (3) is an optional second linker, or spacer
  • Part (4) is a "probe" oligonucleotide which has a
  • a series of sensor arrays each of which comprises a probe oligonucleotide having a different sequence than those of the other sensor arrays, is attached to a common (shared) solid support, although other arrangements can be used.
  • the sensor arrays are arranged in a matrix on a shared solid support.
  • Each "probe” oligonucleotide has a nucleobase sequence that is the precise reverse complement of a corresponding "target" deletion sequence oligonucleotide and is thus capable of specific hybridization with a unique deletion sequence oligonucleotide species. Under appropriate conditions, each target sequence deletion oligonucleotide hybridizes with (binds to) specifically to its corresponding probe oligonucleotide. Applicants have discovered that, for particularly accurate determinations, allowing the hybridization reactions to proceed to equilibrium is preferred. The amount of bound labeled target oligonucleotide bound to a particular sensor array, which correlates directly with the amount of the corresponding deletion sequence oligonucleotide present in the sample, is then determined.
  • deletion sequence oligonucleotides is isolated by means known in the art or explained herein.
  • the sample solution containing full length oligonucleotide and all of the ( ⁇ -1) deletion sequences is loaded onto a polyacrylamide slab gel, and a solution containing primarily or only ( ⁇ -1) components is
  • HPLC or CGE can be used to isolate a sample of ( ⁇ -1) -mers.
  • the relative or absolute amounts of [total deletion sequence oligonucleotides] and [full length oligonucleotide] are determined, either at the same time the sample solution is isolated or by an independent method.
  • the deletion sequence oligonucleotides in the mixture are detectably labeled with, e.g., an enzyme, a fluorescent dye or a radioisotope (e.g., biotin-streptavidin, fluorescein isothiocyanate, 35S, 32P or the like) .
  • an enzyme e.g., an enzyme, a fluorescent dye or a radioisotope (e.g., biotin-streptavidin, fluorescein isothiocyanate, 35S, 32P or the like)
  • a radioisotope e.g., biotin-streptavidin, fluorescein isothiocyanate, 35S, 32P or the like
  • oligonucleotides is contacted to a composition according to the invention.
  • the composition which may be in the form of a matrix, comprises a plurality of sensor arrays, wherein each sensor array comprises a unique oligonucleotide probe that is complementary to only one of the potential deletion sequence oligonucleotides.
  • Each oligonucleotide probe forms a match with its corresponding deletion sequence oligonucleotide and a mismatch with other deletion sequence oligonucleotides present in the mixture.
  • mismatched oligonucleotides hybridize poorly or not at all to the compositions of the invention.
  • the hybridization reactions take place in the absence of electrophoresis or any other flow and are allowed to proceed to equilibrium.
  • oligonucleotides is dependent upon parameters such as the ionic strength of the buffer solution in which the hybridization occurs, temperature, base composition and length of the duplex formed between the target oligonucleotide and the sensor array, concentration of the sensor array, concentration of the target oligonucleotide, and the concentration (s) of duplex destabilizing agent (s) .
  • the method of the invention is designed to maximize the affinity of the probes of the sensor array for the target oligonucleotide while achieving the least degree of affinity for other ( ⁇ -1)
  • SSPE buffer (lx-5x) and 0.1-0.5% SDS; 5x SSPE buffer is 0.75 M NaCl, 50 mM NaH 2 P0 , pH 7.4, and 5 mM EDTA;
  • Temperature can be another important parameter for hybridization reactions.
  • the temperature of the hybridization reaction is adjusted so that only the target oligonucleotide will quantitatively hybridize to the sensor array.
  • the formation of duplexes between the sensor array and undesired oligonucleotides will be thermodynamically disfavored.
  • optimum temperatures can be estimated from the melting temperature ( T m # the temperature at which 50% of
  • the duplex dissociates.
  • the temperature for hybridization reactions should be between the melting temperature of the target oligonucleotide duplex, m ⁇ , and the highest melting
  • T m u temperature of undesired deletion sequence oligonucleotides
  • T temperature range at which the hybridization reaction is performed
  • the temperature at which the hybridization reactions of step (b) occur can be increased
  • the hybridization reactions take place in the absence of electrophoresis or any other flow and are allowed to proceed to equilibrium. Applicants have discovered that, for particularly accurate determinations, allowing the hybridization reactions to proceed to equilibrium is preferred.
  • the methods of the invention optionally comprise the addition of unlabeled target ( ⁇ -1) oligonucleotides during the
  • Step (c) In this optional, but preferred, step, unbound
  • oligonucleotides are removed by washing. Removal of mismatched (undesired) target oligonucleotides, is achieved by placing the hybridized probe: target oligonucleotide complexes into a suitable washing buffer which has a composition that is similar, or even identical, to that of the hybridization buffer of step (b) but which is different in concentration.
  • the washing buffer can be from 0.4x to 2x, preferably from 0.5x to 1.6x, and most preferably from 0.6x to 1.2x the concentration of the hybridization buffer.
  • the washing buffer is from 1.2x to 6x SSPE buffer, preferably from 1.5x to 4.8x SSPE buffer, and most preferably from 1.8x to 3.6x SSPE buffer.
  • the purpose of the optional washing step is to remove as much unbound target deletion sequence oligonucleotide molecules as - Im ⁇
  • Step (d) In this step, the signal intensity of bound
  • label for each sensor array is determined using any appropriate means. For example, radiolabeled oligonucleotides are detected by autoradiography or radiodensitometry, and fluorescently labeled oligonucleotides are detected by measuring the fluorescence present at a given sensor array.
  • Enzymatically labeled oligonucleotides are detected by adding a substrate that undergoes a detectable change (e.g., a chromogenic reaction) that results from the presence of the enzyme. Regardless of the detection means used, the signal intensity is directly proportional to the amount of the specific labeled deletion sequence oligonucleotide bound to a specific sensor array via its particular reverse complementary oligonucleotide probe.
  • a detectable change e.g., a chromogenic reaction
  • each deletion sequence oligonucleotide in the sample solution is determined by comparing the signal intensities of the various sensor arrays.
  • the relative amount of a given deletion sequence oligonucleotide in the sample is multiplied by the total concentration of deletion sequence oligonucleotides in the preparation to yield the absolute concentration of that particular deletion sequence oligonucleotide in the synthetic preparation.
  • the methods of the invention are used to characterize a mixture of ( ⁇ -1) deletion sequence
  • oligonucleotides present in a preparation of synthetic oligonucleotide of length n.
  • a preparation of synthetic oligonucleotide of length n Such a mixture consists of a set
  • nucleobases but each of which has a different nucleobase sequence resulting, in each instance, from the deletion of a single base from the nucleobase sequence of the full-length oligonucleotide.
  • deletion sequence oligonucleotides having differing sequences is hybridized to a composition comprising a variety of probe oligonucleotides.
  • Each probe consists essentially of an oligonucleotide having a nucleobase sequence that is the precise reverse complement of a given ( ⁇ -1) deletion sequence
  • oligonucleotide and a reverse complement probe oligonucleotide is present for every possible ( ⁇ -1) -mer that
  • product of this embodiment of the invention is a determination of the relative and absolute amounts of each ( ⁇ -1) species
  • oligonucleotide intended for therapeutic use.
  • compositions of the invention comprise a solid support (1) to which a plurality of sensor arrays is attached.
  • Each sensor array comprises up to three parts: a first linker to the solid support (2), an optional second linker or spacer
  • Exemplary solid supports (1) include, but are not limited
  • polystyrene or long chain alkyl CPG (controlled pore glass) beads are employed.
  • microscopic glass slides are employed (Fodor et al . , Science, 1991, 251, 767; Maskos et al . , Nucleic
  • the first linker (2) may be selected from a variety of
  • the optional spacer (3) may be employed.
  • a suitable spacer (3) may be employed.
  • linker has the preferred characteristic of non-reactivity with compounds introduced during the various steps of oligonucleotide synthesis. It will be appreciated by those skilled in the art that the chemical composition of the solid support (1), the probe oligonucleotide (4) and, if present,
  • linkers will comprise a primary amine group at either or both termini, as many chemical reactions are known in the art for linking primary amine groups to a variety of other chemical groups; however, other terminal reactive moieties are known and may be used in the invention.
  • Suitable linkers include, but are not limited to, linkers having a terminal thiol group for introducing a disulfide linkages to the solid support (Day et al . , Biochem . J. , 1991, 278, 735;
  • biotin-avidin or biotin-streptavidin linkages (Kasher et al . ,
  • aminoalkyl chain is the linker.
  • oligonucleotide chains constitute both the spacer (3) and the oligonucleotide probe
  • a preferred linker (2) is an ⁇ -
  • aminohexyl chain [i.e., NH 2 -(CH2)g] .
  • the second linker or spacer (3) is optional and may be
  • probe (4) of the sensor array may be employed.
  • a suitable probe (4) of the sensor array may be employed.
  • spacer has the preferred characteristic of non-reactivity with compounds introduced during the various steps of oligonucleotide synthesis. It will be appreciated by those skilled in the art that the chemical composition of the linker (2) and the probe oligonucleotide (4) will influence the linker (2) and the probe oligonucleotide (4).
  • spacer typically suitable spacers include, but are not limited to, oligopeptides ; oligonucleotides; alkyl chains; polyamines; polyethylene glycols; oligosaccharides; and art-recognized equivalents of any of the preceding spacers .
  • the spacer is an alkyl chain, most preferably a C ⁇ -C alkyl chain.
  • the spacer is an oligonucleotide chain, particularly an oligonucleotide chain that comprises one or more chemical modifications that render it resistant to chemical attack.
  • an oligodeoxyribonucleotide chain is particularly preferred.
  • poly(dT)5_3 Q acts as the spacer of the matrix of the invention.
  • This preferred spacer has the following advantages. This spacer is composed of nucleotides and is thus closely related in chemical properties to the preferred sensor array, i.e., an oligonucleotide. This chemical relatedness provides the benefit of placing the sensor array in a context that is likely to be appropriate for nucleic acid hybridization duplexing. Although other polynucleotides [e.g., poly(dA), poly(dG), poly(dC), etc.] might be employed for the spacer, the preferred poly(dT) spacer is more chemically stable.
  • first linker (2) and the second linker or spacer (3) can be any linker (2) and the second linker or spacer (3).
  • linker and spacers need not comprise distinct chemical groups or chains.
  • an appropriate oligopeptide or oligonucleotide chain could function as a combined linker and spacer of the matrix of the invention.
  • suitable linker/spacers include, but are not limited, to the linker and spacers described above. Methods of determining an appropriate linker/spacer length (for, e.g., the purpose of providing the optimal degree and specificity of hybridization between the sensor array and the target oligonucleotide) are known in the art (see, for example, Day et al . , Biochem . J. ,
  • the carbonate moiety is excluded in some instances because it is relatively unstable to reagents used in some oligonucleotide syntheses and to contaminants (mainly bases) that may be found in solvents utilized in some oligonucleotide synthesis.
  • the oligonucleotide probe (4) of a sensor array has a
  • a preferred oligonucleotide probe is one having a nucleobase sequence that is the reverse complement of at least a portion of the nucleobase sequence of the target deletion sequence oligonucleotide.
  • the term "a portion" is intended to encompass at least five contiguous nucleobases uniquely derived from a section of the target deletion sequence oligonucleotide ' s sequence.
  • the oligonucleotide probe is one having a nucleobase sequence that is (a) the reverse complement of the nucleobase sequence of the target deletion sequence oligonucleotide and (b) the same length as that of the target oligonucleotide.
  • a sensor array comprising an oligonucleotide probe having a nucleobase sequence that is the reverse complement of the nucleobase sequence of a target deletion sequence oligonucleotide will hybridize with high affinity to its corresponding target oligonucleotide but not to, e.g., other deletion oligonucleotides having different sequences.
  • an oligonucleotide probe of the sensor array has a sequence that is the "reverse complement" of that of the nucleotide sequence of its target oligonucleotide, the following features are intended.
  • a nucleic acid duplex is formed of two antiparallel strands, i.e., strands that hybridize to each other in a "head-to-tail"
  • nucleobases in the interior of a nucleic acid duplex bind to specific partner nucleobases to form a "base pair" (indicated by a "
  • guanine (G) binds to cytosine (C)
  • adenine (A) binds to thymine (T) or uracil (U) .
  • Strand 2 will have a nucleotide sequence that is the reverse complement of Strand 1, i.e.,
  • Strand 2 will have, in "reverse” (3' to 5 ' ) order, the partner ("complement") nucleobases to those of Strand 1.
  • the sequence of the oligonucleotide of the sensor array can have reverse complementarity to the target oligonucleotide through a variety of equivalents.
  • other naturally occurring nucleobase equivalents including 5-methylcytosine (m5c) , 5-hydroxymethylcytosine (HMC) (C equivalents) and 5-hydroxymethyluracil (U equivalent) .
  • synthetic nucleobases which retain partner specificity are known in the art and include, for example, 7-deazaguanine, which retains specificity for C and is thus a G equivalent.
  • reverse complementarity will not be altered by any chemical modification to a nucleobase in the nucleotide sequence of the affinity oligonucleotide which does not alter its specificity for the partner nucleobase in the target oligonucleotide.
  • probe oligonucleotides present in a given composition depends on the intended use for the
  • composition and the nature of the sequence of the full-length "parent" oligonucleotide For example, for the characterization of ( ⁇ -1) -mers present in a synthetic preparation of a "parent" oligonucleotide of length ⁇ , the
  • oligonucleotides that have the same sequence. For example, for the parent sequence
  • G-G-C-T-T-T-C (deletion at positions 4-7) G-G-C-T-T-T (deletion at position 8)
  • the nucleobase sequence of the oligonucleotide probe of the sensor array can be from 5 to about 50 nucleotides in length, preferably from 6 to about 25 nucleotides in length, more preferably from 8 to about 15 nucleotides in length.
  • oligonucleotide probes of differing chemical compositions e.g., oligodeoxynucleotides,
  • oligoribonucleotides and peptide nucleic acids can be employed in the invention, peptide nucleic acids and oligodeoxyribonucleotides are preferred in particular instances for the following reasons. Unlike RNA nucleases, for which no "universal" inhibitor is known, all characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents such as EDTA (Jarrett, J " . Chroma togr. , 1993, 618 , 315); oligodeoxyribonucleotides can thus be more simply prevented from degradation than oligoribonucleotides. Peptide nucleic acids exhibit particularly stringent specificities for their complementary oligonucleotides, and may thus provide the best degree of separation from undesired derivative oligonucleotides in some instances.
  • the oligonucleotide of the sensor array can incorporate one or more chemical modifications for the purpose of enhancing specific interactions with the target oligonucleotide. Such modifications may additionally or alternatively result in the oligonucleotide of the sensor array having increased resistance to degradative contaminants, e . g. , exonucleases .
  • the target oligonucleotides may additionally or alternatively comprise such modifications, so long as reverse complementarity is maintained between the sequence of the target oligonucleotide and that of the probe oligonucleotide of the sensor array.
  • Components of an oligonucleotide that can be modified include the sugar (ribofuranosyl) portion, the nucleobase portion and one or more of the chemical linkages that make up an oligonucleotide ' s backbone. Specific chemical modifications of particular interest are described in the Examples .
  • linker, spacer and probe oligonucleotide can be combined into one structurally linked unit.
  • the linker, spacer and probe oligonucleotide need not comprise distinct chemical groups or chains.
  • an oligonucleotide of appropriate chain length and sequence could function as the linker, spacer and probe oligonucleotide of a sensor array.
  • the spacer and probe oligonucleotide of a sensor array can be combined into one unit.
  • probe oligonucleotide and spacer need not comprise distinct chemical groups or chains.
  • an aminohexyl group is the linker to the solid support, as it is easily attached to the 5' end of a oligonucleotide by a solid phase synthesizer.
  • the probe oligonucleotide of a sensor array extends beyond its probe sequence (i.e., the sequence having reverse complementarity to all or a portion of a target deletion sequence oligonucleotide) to include a further nucleotide sequence which functions as the spacer of the sensor array.
  • Sensor arrays may be attached to the solid support by chemical conjugation of pre-synthesized sensor arrays to the support .
  • the sensor array is synthesized directly on the solid support (i.e., in si tu)
  • the number and sequences of different probe oligonucleotides present in a given composition depends on the intended use for the composition and the sequence of the parent oligonucleotide from which target deletion sequence oligonucleotides are derived.
  • a synthetic oligonucleotide known as ISIS 2922 was chosen as an exemplary parent oligonucleotide for a series of experiments involving the methods and compositions of the invention.
  • ISIS 2922 is a synthetic 21 base ( ⁇ -mer) antisense
  • oligonucleotide targeted to cytomegalovirus having the following sequence (see SEQ ID NO: 22 in U.S. Patent No. 5,442,049) :
  • oligonucleotide sequences in this example than are possible for a 21-mer devoid of any such contiguous and identical residues .
  • Each oligonucleotide probe only hybridizes with the corresponding target oligonucleotide of ( ⁇ -1) deletion sequence through
  • the probes are natural or derivative oligonucleotides having a length of 20 bases.
  • Each of the probes used in the Examples of the present disclosure includes a 3 ' -terminal eight base sequence that is the reverse complement of the most 5 ' eight bases of a specific target ( ⁇ -1) oligonucleotide (Table 2) .
  • Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry with oxidation using iodine. Beta- cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, CA) . For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1, 2-benzodithiole-3-one-l , 1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages .
  • the 2 ' -fluoro-phosphorothioate oligonucleotides of the invention are synthesized using 5 ' -dimethoxytrityl-3 ' - phosphoramidites and prepared as disclosed in U.S. patent application Serial No. 08/383,666, filed February 3, 1995, and U.S. Patent 5,459,255, which issued October 8, 1996, both of which are assigned to the same assignee as the instant application and which are incorporated by reference herein.
  • the 2 ' -fluoro-oligonucleotides are prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol (i.e., deprotection was effected using methanolic ammonia at room temperature) .
  • PNA antisense analogs are prepared essentially as described in U.S. Patents Nos. 5,539,082 and 5,539,083, both of which (1) issued July 23, 1996, (2) are assigned to the same assignee as the instant application and (3) are incorporated by reference herein.
  • Oligonucleotides comprising 2 , 6-diaminopurine are prepared using compounds described in U.S. Patent No. 5,506,351 which issued April 9, 1996, and which is assigned to the same assignee as the instant application and incorporated by reference herein, and materials and methods described by Gaffney et al . ⁇ Tetrahedron, 1984, 40:3), Chollet et al . ,
  • Oligonucleotides comprising 2,6- diaminopurine can also be prepared by enzymatic means (Bailly et al . , Proc . Na tl . Acad. Sci . U. S . A . , 1996, 93:13623) .
  • the 2 ' -methoxyethoxy oligonucleotides of the invention were synthesized essentially according to the methods of Martin et al . ⁇ Helv. Chim . Acta , 1995, 78, 486).
  • the 3' nucleotide of the 2 ' -methoxyethoxy oligonucleotides was a deoxynucleotide, and 2 ' -0-CH 2 CH 2 OCH 3 .
  • cytosines were 5-methyl cytosines, which were synthesized according to the procedures described below.
  • 5-methyluridine 2 ' -O-Methoxyethyl-5 ' -O-dimethoxytrityl-5- methyluridine (106 g, 0.167 M) , DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by thin layer chromatography (tic) by first quenching the tic sample with the addition of MeOH. Upon completion of the reaction, as judged by tic, MeOH (50 mL) was added and the mixture evaporated at 35 C.
  • tic thin layer chromatography
  • 5-methyl-4-triazoleuridine A first solution was prepared by dissolving 3 ' -0-acetyl-2 ' -0-methoxyethyl-5 ' -O-dimethoxytrityl- 5-methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L) , cooled to -5 C and stirred for 0.5 h using an overhead stirrer.
  • the dioxane solution was evaporated and the residue azeotroped with MeOH (2x 200 mL) .
  • the residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel.
  • Methanol (400 mL) saturated with NH 3 gas was added and the vessel heated to 100 C for 2 hours (thin layer chromatography, tic, showed complete conversion) .
  • the vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL) .
  • the organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.
  • N-Benzoyl-2 ' -O-methoxyethyl-5 ' -O-dimethoxytrityl-5- methylcytidine 2 ' -O-Methoxyethyl-5 ' -O-dimethoxytrityl-5- methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tic showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL) .
  • the ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL) .
  • the solution was poured into fresh ether (2.5 L) to yield a stiff gum.
  • the ether was decanted and the gum was dried in a vacuum oven (60°C at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield) .
  • the NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%) .
  • 5-methyluridine 2 ' -O-Methoxyethyl-5 ' -0-dimethoxytrityl-5- methyluridine (106 g, 0.167 M) , DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tic by first quenching the tic sample with the addition of MeOH. Upon completion of the reaction, as judged by tic, MeOH (50 mL) was added and the mixture evaporated at 35°C.
  • 5-methyl-4-triazoleuridine A first solution was prepared by dissolving 3 ' -0-acetyl-2 ' -0-methoxyethyl-5 ' -O-dimethoxytrityl- 5 -methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L) , cooled to -5°C and stirred for 0.5 h using an overhead stirrer.
  • POCl 3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10°C, and the resulting mixture stirred for an additional 2 hours.
  • the first solution was added dropwise, over a 45 minute period, to the later solution.
  • the resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed once with 300 mL of NaHC0 3 and 2x 300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.
  • N 4 -Benzoyl-2 ⁇ -0-methoxyethyl-5 ' -O-dimethoxytrityl-5- methylcytidine 2 ' -O-Methoxyethyl-5 ' -O-dimethoxytrityl-5- methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tic showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL) .
  • the resulting mixture was stirred for 20 hours at room temperature (tic showed the reaction to be 95% complete) .
  • the reaction mixture was extracted with saturated NaHC0 3 (lx 300 mL) and saturated NaCl (3x 300 mL) .
  • the aqueous washes were back- extracted with CH 2 C1 2 (300 mL) , and the extracts were combined, dried over MgS0 4 and concentrated.
  • the residue obtained was chromatographed on a 1.5 kg silica column using
  • Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per the methods of United States patent applications serial number 10/037,143, filed February 14, 1998, and serial number 09/016,520, filed January 30, 1998, each of which is commonly owned with the instant application and is herein incorporated by reference.
  • the thiation wait step was increased to 68 sec and was followed by the capping step.
  • the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.
  • Phosphinate oligonucleotides are prepared as described in U.S. Patent 5,508,270, herein incorporated by reference.
  • Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference .
  • 3 ' -Deoxy-3 ' -methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, herein incorporated by reference.
  • Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, herein incorporated by reference.
  • Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively) , herein incorporated by reference.
  • 3 ' -Deoxy-3 ' -amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, herein incorporated by reference.
  • Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, herein incorporated by reference .
  • Borano phosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, both herein incorporated by reference.
  • Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Patents 5,264,562 and 5,264,564, herein incorporated by reference.
  • Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference .
  • Example 3 Coupling the Solid Support (1) to a Linker (2)
  • Glass material is preferred as a solid support for the probes .
  • Commercial glass is a rigid liquid product of fusion with inorganic ingredients.
  • Silica (Si02) derived from sand provides the structure backbone. There are Si-OH bonds on the glass surface.
  • Both microscopic glass slides and derivatized controlled-pore glass can be used as the solid support to attach the modified oligonucleotide probe.
  • the glass surface is modified to form an amino-reactive terminal.
  • the modification can be achieved by treating the glass surface (GS) with a chemical, (CH 3 0) 3 Si (CH 2 ) 3 NCS :
  • Another way to form the amino-active terminal on the glass surface is to use two step reactions. The first reaction forms a primary amine, and then in the second reaction, this primary amine reacts with 1, 4-phenylene diisothiocyanate to form the amino reactive phenylisothiocyanate group.
  • amino- propyl CPG or long chain amino CPG is preferred.
  • the primary amine is located on the glass surface and modification is achieved by reacting this amino group of the glass surface with 1, 4-phenylene diisothiocyanate to form the amino reactive phenylisothiocyanate group:
  • the [probe oligonucleotide/spacer] unit of the sensor array are labeled with moieties which can produce detectable signals. Labeling can be achieved by attaching a fluorescent dye, or a radioisotope such as 32P or
  • Fluorescent labeling can be achieved by reacting a commercially available nucleoside terminator labeled with a dye (for example, carboxyfluorescein (FAM)) such as, for example, ddC-5FAM, with the probe (in the ⁇ py following example reactions, probe P9 , SEQ ID NO: 30) in the presence of an enzyme, deoxynucleotidyl transferase:
  • a dye for example, carboxyfluorescein (FAM)
  • FAM carboxyfluorescein
  • Radioisotope Labeling can • 3K OO be achieved by reacting [cc- S]dATP, or [cc- P] dATP with the probe (in the following example reactions, probe P9 FX , SEQ ID NO: 30) in the presence of the same enzyme:
  • Oligonucleotides may also be labeled with enzymatic groups, or groups that bind an enzyme (e.g., biotin) , and subsequently detected by chemical reactions catalyzed by such enzymes.
  • an enzyme e.g., biotin
  • a variety of enzyme- oligonucleotide conjugates, and means of preparing such conjugates, are known in the art (see, e.g., Ruth, Chapter 6 In : Methods in Molecular Biology, Vol . 26 : Protocols for
  • Target (n-1) Oligonucleotides The target oligonucleotides of (n-1) deletion sequences can be labeled with fluorescent dye or radioisotope according to the following example reactions.
  • target oligonucleotide Dll SEQ ID NO: 12
  • target ( ⁇ -1) oligonucleotide is used as the target ( ⁇ -1) oligonucleotide.
  • Fluorescent labeling can be achieved by reacting a commercially available dye labeled nucleoside terminator, ddC-5FAM with the target oligonucleotide in the presence of deoxynucleotidyl transferase :
  • Radioisotope Labeling The target oligonucleotides of ( ⁇ -1) deletion sequence oligonucleotides are labeled with radioisotope (such as, for example, 35g or 32p) at either the 3' end with deoxynucleotidyl transferase or the 5' end with T4 nucleotidyl kinase .
  • radioisotope such as, for example, 35g or 32p
  • Example 4 For each sample, 2 uL of labeled probe, at a
  • Hybridization the formation of a double helix from two oligonucleotides, is a reversible process. Hybridization is dependent upon ionic strength, base composition, the length of the double helix, the concentration of the probe, the concentration of target oligonucleotides and the concentration of helix destabilizing agents.
  • the stability of a duplex formed between strands with mismatched bases is affected by the number and location of mismatches. For oligonucleotides, the T m decreases by approximately 5 C for every mismatched base pair. The greater number of mismatches, the easier the sequence discrimination between the matched and imperfectly matched oligonucleotides. The middle position of the mismatch is preferred for better differentiation.
  • Hybridization stringency can be adjusted by varying the salt concentration, the concentration of destablizing agents such as SDS and/or formamide, and/or by changing the temperature of the hybridization reactions .
  • the degree of discrimination can also be enhanced by adjusting the post hybridization washes. For example, the hybridization can be performed at low stringency and washed a number times, using either the same elution solution or different ones with the increasing stringencies, and the signal intensity measured after each wash.
  • Probe Concentration The relative concentration of the probes present in the sensor array is another factor affecting the degree of selectivity for specific target ( ⁇ -1) deletion oligonucleotides. The probe concentration should be optimized for specific hybridization of the corresponding matched target oligonucleotide thereto.
  • Table 5 lists the relative intensity of target oligonucleotides D3 and D14 (SEQ ID NOS: 4 and 15, respectively; 0.5 pmol/uL) hybridized to their corresponding matching probes P3 and P14 (SEQ ID NOS: 18 and 29, respectively) at different probe concentrations (0 to 7 pmol/uL) for three (3) hours.
  • the relative intensity increases as the probe concentration increases in the 0-0.1 pmol/uL range and remains nearly constant in the 0.1-7.0 pmol/uL range.
  • a probe concentration of 0.5 pmol/uL is preferred. Higher probe concentrations may result in nonspecific binding of mismatched oligonucleotide.
  • Target Oligonucleotide Concentration Another factor affecting the degree of selectivity for specific target ( ⁇ -1) deletion oligonucleotides is the relative concentration of the target ( ⁇ -1) deletion oligonucleotides.
  • the target oligonucleotide concentration should be optimized for specific hybridization to the corresponding matched oligonucleotide probe.
  • Target oligonucleotide D3 (SEQ ID NOS: 4) was labeled with [ ⁇ - 35S]dATP using T4 nucleotidyl kinase as described in
  • Example 5 shows the relative intensity of different concentrations (0.1 to 4 pmol) of target oligonucleotide D3 (SEQ ID NO: 4) hybridized to its corresponding matching probe P3 (SEQ ID NO: 18) at three probe concentrations (0.2, 1 and 2 pmol) for three (3) hours.
  • the data in Table 6 show that there is a linear relationship between the signal intensity and the target oligonucleotide concentration when the probe is not saturated. That is, a relatively high concentration of probe (2 uL of 1 pmol/uL) has better linearity for the quantitation of the hybridization reactions.
  • D. Effect of Temperature Another factor affecting nucleic acid hybridization reactions is temperature.
  • target oligonucleotides D3 (SEQ ID NO: 4) and D14 (SEQ ID NO: 15) were labeled as in Example 5 and hybridized to their cognate probe oligonucleotides, P3 (SEQ ID NO-.18) and P14 (SEQ ID NO:29), respectively, at various temperatures (30 to 50 C) for three (3) hours.
  • the relative intensity of hybridized material as determined by scanning the autoradiograph of the reactions on a 300S Molecular Dynamics densitometer is shown in Table 7. The results demonstrate that selectivity increases with increasing temperature until about 45 C. As the temperature rises to greater than 50 C, the melting temperature (T m ) of the perfectly matched duplex is exceeded, resulting in a decrease in hyrbidization efficiency. In this instance, the optimum temperature range for the hybridization reactions is
  • Unlabeled or "cold" target oligonucleotide can be used to suppress hybridization of labeled or "hot” target oligonucleotide to a mismatched probe.
  • D3 cold target oligonucleotide
  • D14 D14
  • Both target oligonucleotides were hybridized to probe P14 (SEQ ID NO: 29) which is complementary to D14 but which mismatched to D3.
  • Cold D3 was added over a concentration range of 0 to 10 pmol to both hybridization reactions (i.e., D14 : P14 and
  • % interference i.e., the ratio, expressed as a percentage, of the relative intensity of the mismatched D3 : P14 duplex to that of the perfectly matched D14:P14 duplex.
  • the results demonstrate that the interference by labeled target oligonucleotide D3 (mismatched) of the matched D14:P14 hybridization reaction was reduced from 25% to 5% by the addition of 10 pmol/uL of unlabeled target oligonucleotide D3 (Table 8) .
  • a matrix of sensor arrays comprising unlabeled probes Pl to P14 (SEQ ID NOS: 16 to 29) was constructed on a glass slide according to the method of Examples 1 and 2.
  • target ( ⁇ -1) oligonucleotides D5 (SEQ ID NO:4) and D7 (SEQ ID NO: 6) were 5' end-labeled with 35 S and T4 nucleotidyl kinase according to the methods described in Example 5.
  • the target oligonucleotides were hybridized to separate sensor arrays.
  • the microscope slides comprising the sensor arrays were washed with 2x SSPE buffer solution for 20 minutes and dried.
  • the sensor arrays, to which labeled target oligonucleotides were hybridized, were exposed to AIF film (Fisher Scientific, Pittsburgh, PA) which was then developed according to methods well-known in the art.
  • the autoradiographs were scanned by a densitometer (Molecular Dynamics, Sunnyvale, CA) .
  • target oligonucleotides D5 SEQ ID NO: 4
  • D7 SEQ ID NO: 6
  • Table 9 the relative intensity of hybridization signal is indicated for probes Pl to P14.
  • the selectivity in specific hybridization is reflected in the fact that the clear majority of target oligonucleotide D5 hybridizes to matched probe P5 (SEQ ID NO: 20) .
  • the clear majority of target oligonucleotide D7 hybridizes to matched probe P7 (SEQ ID NO: 22) .
  • lots "A” and "B" of ISIS 2922 SEQ ID NO:l were evaluated by the methods and compositions of the disclosure in order to examine the invention's ability to evaluate the composition of ( ⁇ -1) target oligonucleotide compositions from different ( ⁇ -mer) oligonucleotide syntheses.
  • the ( ⁇ -1) target oligonucleotide populations were isolated from the oligonucleotide BDS (bulk drug substance) by cutting the ( ⁇ -1) band of each lot out of a polyacrylamide gel and then subjecting the isolated bands to freezing and thawing.
  • the ( ⁇ -1) mixture was further purified and concentrated by ethanol/acetate precipitation according to methods known in the art.
  • the ( ⁇ -1) target oligonucleotide populations were 5' end-labeled with [ ⁇ - 35S] dATP and T4 nucleotidyl kinase according to the methods described in
  • Example 5 and hybridized to the sensor array comprising probes Pl to P14 SEQ ID NOS: 16 to 29; see Examples 1 and 7) .
  • the matrix and method of the invention detected significant differences in the relative amounts of the indicated ( ⁇ -1) deletion products.
  • lot “A” has a relative intensity of 2.72 for ( ⁇ -1) target oligonucleotide D5 (SEQ ID NO:4)
  • lot “B” has a relative intensity of 1.91 for D5.
  • lot “A” has a relative intensity of 0.58 for ( ⁇ -1) target oligonucleotide D8 (SEQ ID NO:9)
  • lot “B” has a relative intensity of 0.96 for D8.
  • Absolute concentrations can be determined, for example, by the following method.
  • the total amount of nucleic acid material (i.e, both ⁇ -mer and ( ⁇ -1) oligonucleotides, as well as other deletion sequences) in the oligonucleotide preparation is determined by means well known in the art such as, for example, measuring the optical density of the preparation in a spectrometer at an O.D. (optical density) of 260 nm and converting the results to concentrations according to known formulas.
  • O.D. optical density
  • the relative amount of ( ⁇ -1) material in the preparation is determined by methods known in the art such as, for example, densitometric scanning an autoradiograph of a radiolabeled sample of the preparation that has been electrophoresed on an acrylamide, or measuring the OD 260 of aliquots of an unlabeled sample of the preparation that have been separated by HPLC (high performance liquid chromatography) or CGE (capillary gel electrophoresis) .
  • HPLC high performance liquid chromatography
  • CGE capillary gel electrophoresis
  • RA D5 represents the relative amount of ( ⁇ -1) oligonucleotide D5 in the ( ⁇ -1) subpopulation
  • ( n- l) represents the relative amount of all ( ⁇ -1) oligonucleotides in the ⁇ -mer preparation
  • [OLI] represents the absolute amount of all oligonucleotides in the ⁇ -mer preparation
  • [D5] represents the absolute amount of ( ⁇ -1) oligonucleotide D5 in the n-mer preparation.
  • the invention relates to compositions and methods for the identification and characterization of (n-1) deletion sequence oligonucleotides in a mixture comprising a synthetic oligonucleotide of length n.
  • the synthetic oligonucleotide of length n has biological activity and is designed to be administered to cultured cells, isolated tissues and organs and animals.
  • biological activity it is meant that the oligonucleotide functions to modulate the expression of one or more genes in cultured cells, isolated tissues or organs and/or animals.
  • Such modulation can be achieved by an antisense oligonucleotide by a variety of mechanisms known in the art, including but not limited to transcriptional arrest; effects on RNA processing (capping, polyadenylation and splicing) and transportation; enhancement of cellular degradation of the target nucleic acid; and translational arrest (Crooke et al . , Exp . Opin . Ther. Patents, 1996, 6:855) .
  • compositions and methods of the invention can be used to study the function of one or more genes in the animal.
  • antisense oligonucleotides have been systemically administered to rats in order to study the role of the N-methyl-D-aspartate receptor in neuronal death, to mice in order to investigate the biological role of protein kinase C- ⁇ , and to rats in order to examine the role of the neuropeptide Yl receptor in anxiety (Wahlestedt et al . , Nature, 1993, 363:260; Dean et al . , Proc . Na tl . Acad . Sci . U. S . A . , 1994, 51:11762; and
  • antisense knockouts i.e., inhibition of a gene by systemic administration of antisense oligonucleotides
  • antisense oligonucleotides may represent the most accurate means for examining a specific member of the family (see, generally, Albert et al . , Trends
  • compositions and methods of the invention also have therapeutic uses in an animal, including a human, having (i.e., suffering from), or known to be or suspected of being prone to having, a disease or disorder that is treatable in whole or in part with one or more nucleic acids.
  • therapeutic uses is intended to encompass prophylactic, palliative and curative uses wherein the oligonucleotides of the invention are contacted with animal cells either in vivo or ex vivo .
  • a therapeutic use includes incorporating such cells into an animal after treatment with one or more oligonucleotides of the invention.
  • oligonucleotides can be useful therapeutic instrumentalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
  • U.S. patents demonstrate palliative, therapeutic and other methods utilizing antisense oligonucleotides.
  • U. S. Patent No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression.
  • U.S. Patent No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions.
  • U.S. Patent No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides.
  • U.S. Patent No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication.
  • U.S. Patent No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenza virus.
  • Patent No. 4,806,463 provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication.
  • U.S. Patent No. 5,286,717 provides oligonucleotides having a complementary base sequence to a portion of an oncogene.
  • U.S. Patent No. 5,276,019 and U.S. Patent No. 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells.
  • U.S. Patent No. 4,689,320 is directed to antisense oligonucleotides as antiviral agents specific to cytomegalovirus (CMV) .
  • CMV cytomegalovirus
  • 5,098,890 provides oligonucleotides complementary to at least a portion of the mRNA transcript of the human c- myb gene.
  • U.S. Patent No. 5,242,906 provides antisense oligonucleotides useful in the treatment of latent Epstein- Barr virus (EBV) infections.
  • EBV Epstein- Barr virus
  • Such desired oligonucleotides include, but are not limited to, those designed to modulate cellular adhesion (Table 11) .
  • Other oligonucleotides are designed to modulate cellular proliferation (Table 12), or to have biological or therapeutic activity against miscellaneous disorders (Table 13) and diseases resulting from eukaryotic pathogens (Table 14) , retroviruses including HIV (human immunodeficiency virus; Table 15) or non-retroviral viral viruses (Table 16) . Further details regarding the sources of the following oligonucleotides are provided in the Sequence Listing.
  • ID NO(S) Molecular Name (if any) ID NO(S) :
  • CMV Virus cytomegaloviru GEM 132 122 s
  • target oligonucleotides are "DNA-like” (i.e., having 2 ' -deoxy sugars and T rather than U bases) or "RNA-like” (i.e., having 2 ' -hydroxyl or 2 ' -modified sugars and U rather than T bases) .
  • an oligonucleotide can comprise a majority of 2 ' -deoxy sugars and a few 2'- hydroxyl sugars and still be considered "DNA-like" for the purposes of this invention.
  • some probe oligonucleotide chemistries are preferred for the characterization of DNA-like target oligonucleotides, some are preferred for RNA-like target oligonucleotides, and some function with approximately equal effectiveness for either type of target oligonucleotide.
  • DNA-like target oligonucleotides include but are not limited to oligonucleotides that are entirely or predominately oligodeoxynucleotides (ODNs; i.e., 2 ' -deoxy- oligonucleotides) , and/or have the oxygen of the furanosyl group replaced with S or CH 2 , and/or have one or more base modifications such as, e.g., 5-methylcytosine (m5c) in lieu of cytosine (C) ; 2 , 6-diaminopurine (DAP, also known as 2- aminoadenine) in lieu of adenine (A) ; 2-aminoguanine in lieu of guanine (G) ; and hypoxanthine (I) in lieu of any other nucleobase.
  • ODNs oligodeoxynucleotides
  • m5c 5-methylcytosine
  • C cytosine
  • DAP 6-diamin
  • Preferred probe oligonucleotides for the hybridization of DNA-like target oligonucleotides are generally other DNA-like oligonucleotides, including but not limited to oligonucleotides having (1) a fully or predominantly phosphodiester backbone, (2) entirely or predominately 2'- deoxy-oligonucleotides, (3) the oxygen of the furanosyl group replaced with S or CH 2 , and/or (4) one or more base modifications.
  • Such base modifications include, for example, 2 , 6-diaminopurine (DAP, also known as 2-aminoadenine) in lieu of adenine (A) ; 2-aminoguanine (2AG) in lieu of guanine (G) ; hypoxanthine (I) in lieu of any other nucleobase; 5- (1- propynyl) uracil (5PU) in lieu of thymine (T) ; and 5- (1- propynyl) cytosine (5PC) or 5-methylcytosine (m5c) in lieu of cytosine (C) .
  • DAP 6-diaminopurine
  • 2-aminoadenine 2-aminoguanine
  • 2AG 2-aminoguanine
  • G guanine
  • I hypoxanthine
  • I in lieu of any other nucleobase
  • T thymine
  • 5PC 5- (1- propynyl
  • Probe oligonucleotides having these preferred chemical modifications are synthesized according to the methods and teachings incorporated by reference set forth in Example 2 and in Prosnyak et al . ⁇ Genomics, 1994, 21 , 490; DAP and m5c) ,
  • RNA-like target oligonucleotides include but are not limited to oligonucleotides that are entirely or predominately oligoribonucleotides (i.e., 2 ' -hydroxy- oligonucleotides) , oligonucleotides having a majority of sugars with 2' modifications, and oligonucleotides having a fully or predominately phosphorothioate backbone.
  • Preferred probe oligonucleotide chemistries for the hybridization of RNA-like target oligonucleotides have been extensively described by Freier et al . ⁇ Nucleic Acids Research,
  • Such preferred probe oligonucleotide chemistries include but are not limited to sugar modifications [such as 2'-fluoro; 2'-0-alkyl; 2 ' -methoxyethoxy; 2'-propyl-0- butyl; 2 '- (ethylene glycol) 2 _ 4 ; 2'-nonyl; 2'- dimethylaminoethoxy; 2 ' -dimethylamino-ethoxyethoxy; 2'- monomethylaminoethoxy; 2 ' -aminoethoxy; 2 ' -piperazinethoxy; 2 ' - (3 ' -N,N-dimethylamino-l-propyl) aminoethoxy; and 2 ' -0-CH 2 -CHR- X, where X is OH, F, CF 3 or 0CH 3 and R is independently H, CH 3 , CH 2 0H or CH 2 OCH 3 ] , modified
  • Probe oligonucleotides having these preferred chemical modifications are synthesized according to the methods and teachings incorporated by reference set forth in Example 2 and in published PCT application WO 97/46569, European Patents 0626387 and 0679657, and in copending U.S. patent applications having Serial Nos. 09/115 , 025 (Attorney Docket No. ISIS-2951 filed July 14, 1998); 09/115,027 (Attorney Docket No. ISIS- 2953 filed July 14, 1998): 09/066,638 (Attorney Docket No. ISIS-2914 filed April 24, 1998); 60/078,637 (Attorney Docket No.
  • PNAs Peptide nucleic acids
  • a single composition having PNA probes in its sensor arrays can be used, for example, to characterize the ( ⁇ -1) deletion sequence oligonucleotides present in a preparation of a synthetic hybrid oligonucleotide that comprises both DNA-like and RNA- like portions.
  • Probe peptide nucleic acids are synthesized according to the methods set forth in Example 2.

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Abstract

L'invention porte sur des compositions et procédés d'identification et de quantification d'un mélange de différents oligonucléotides à séquence de délétion présents dans une préparation d'oligonucléotides de synthèse de longueur n, au moyen de réactions d'hybridation.
PCT/US1998/018084 1997-09-02 1998-09-01 Compositions et procedes d'identification et de quantification d'oligonucleotides a sequence de deletion dans des preparations d'oligonucleotides de synthese WO1999011820A1 (fr)

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SOUTHERN E. M., MASKOS U., ELDER J. K.: "ANALYZING AND COMPARING NUCLEIC ACID SEQUENCES BY HYBRIDIZATION TO ARRAYS OF OLIGONUCLEOTIDES: EVALUATION USING EXPERIMENTAL MODELS", GENOMICS, ACADEMIC PRESS, SAN DIEGO., US, vol. 13., 1 January 1992 (1992-01-01), US, pages 1008 - 1017., XP002913497, ISSN: 0888-7543, DOI: 10.1016/0888-7543(92)90014-J *

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US7013221B1 (en) 1999-07-16 2006-03-14 Rosetta Inpharmatics Llc Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays
US7371516B1 (en) 1999-07-16 2008-05-13 Rosetta Inpharmatics Llc Methods for determining the specificity and sensitivity of oligonucleo tides for hybridization
WO2001046214A3 (fr) * 1999-12-21 2001-12-27 Lion Bioscience Ag Compose comprenant un groupe fonctionnel d'acide nucleique et un groupe fonctionnel silane organique
US7807447B1 (en) 2000-08-25 2010-10-05 Merck Sharp & Dohme Corp. Compositions and methods for exon profiling
US7262031B2 (en) 2003-05-22 2007-08-28 The Regents Of The University Of California Method for producing a synthetic gene or other DNA sequence

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