WO2001002558A1 - Probe for analysis of nucleic acids - Google Patents

Probe for analysis of nucleic acids Download PDF

Info

Publication number
WO2001002558A1
WO2001002558A1 PCT/SE2000/001429 SE0001429W WO0102558A1 WO 2001002558 A1 WO2001002558 A1 WO 2001002558A1 SE 0001429 W SE0001429 W SE 0001429W WO 0102558 A1 WO0102558 A1 WO 0102558A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
group
sre
carbon atoms
alkyl
Prior art date
Application number
PCT/SE2000/001429
Other languages
French (fr)
Other versions
WO2001002558A8 (en
Inventor
Jonas Karlsson
Gunnar Westman
Original Assignee
Lightup Technologies Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lightup Technologies Ab filed Critical Lightup Technologies Ab
Priority to AU60427/00A priority Critical patent/AU6042700A/en
Publication of WO2001002558A1 publication Critical patent/WO2001002558A1/en
Publication of WO2001002558A8 publication Critical patent/WO2001002558A8/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the invention belongs to the category probes for hybridization to nucleic acids, and in particular to fluorescence dyes used in such probes.
  • Such probes are used in methods where specific genes, gene segments, RNA molecules and other nucleic acids are identified. These methods are primarily used clinically, for example to test tissue, blood and urine samples, in food technology, agriculture and in biological research.
  • a further object is to obtain fluorescent dyes that differs between DNA and PNA when attached to a probe.
  • probes of toady One deficit belonging to the probes of toady is that they, to a certain degree are self- fluorescent, even when they do not bind to the DNA, so called background fluorescence, which results in a bad sensitivity.
  • background fluorescence By developing fluorescent dyes which bind to DNA only and/or only bind to the complex that DNA and nucleic acid of the probe forms it is possible to provide a more sensitive probe.
  • NA nucleic acids
  • RNA ribonucleic acids
  • a probe based on an oligodeoxyribonucleotide equipped with a reporter group (RG) that usually is a radioisotope employs usually the following steps: the nucleic acid to be tested is immobilized on a paper, glass bead or plastic surface; an excess of probe complementary to the target sequence is added; the probe is allowed to hybridize; non- hybridized probe is removed; remaining probe bound to the immobilized target sequence is detected.
  • RG reporter group
  • Non-hybridized probe is removed by extensive washing. This is usually the most time consuming and critical step in the procedure. Since the properties of non-hybridized and hybridized probe are not distinguishable, it is necessary that essentially all non-hybridized probe is removed. Since the hybridized probe is only attached through its interaction with the target sequence also some of it will be removed by washing, as well as some hybrids between TS and probe where TS was not sufficiently immobilized. Further, some probe may stick directly to the surface giving rise to a background signal. Finally, the requirement that non-hybridized probe must be removed makes in vivo and real time detection impossible.
  • Bannwarth et al. (Helvetica Chimica Acta, 71, 2085, 1988) have developed a method with probes composed of an oligodeoxyribonucleotide equipped with a ruthenium complex, where hybridization can be demonstrated from measurements of the probe fluorescence lifetime.
  • the strategy is elegant, its application is limited to specialized laboratories that have sophisticated instrumentation, and can only be used by people with special training. Further, the ability of the method to distinguish hybridized and non- hybridized probe is not too good, particularly not in biological samples that may contain components that affect the probe fluorescence life time.
  • Barton J. (US Pat. 5.157.032) describes a probe composed of a DNA-chain modified with a metal-ligand complex whose fluorescence intensity increases upon hybridization. These probes obtain only a modest fluorescence upon hybridization (a fluorescence quantum yield of 0.007 has been reported, Jenkins & Barton, J. Am. Chem. Soc, 114, 8736, 1992), which gives low sensitivity. Further, the probes are dicationic (charge +2), which leads to considerable non-specific contribution to the interaction and consequently a decreased ability to distinguish different sequences.
  • Yamana et al. (Nucl. & Nucl. 11 (2-4), 383, 1992) describe a probe composed of an oligonucleotide modified with pyrene, which under optimal conditions gives a 20-fold increase in fluorescence upon hybridization.
  • the method has several disadvantages. Pyrene has complicated photophysics and its absorption and fluorescence properties depend on its closest surrounding; for example, it has a large tendency to form excimers (J. Michl & Erik W. Thulstrup in Spectroscopy with polarized light, 1 st Ed. VCH, 1986, ISBN 0-89573-346- 3). Further, pyrene emits ultraviolet light (below 450 am) that cannot be seen by the naked eye. Finally, pyrene is toxic (Yoshikawa et al, Vet. Hum. Toxicol. 29, 25, 1987).
  • Heller et al (EPA 070685) and Cardullo et al., (Proc. Natl. Acad. Sci. USA, 85, 8790-8794, 1988) describe a probe based on simultaneous hybridization of two DNA-based probes to close-lying sequences.
  • One probe is modified in the 3 '-terminus of the DNA chain with a donor fluorophore and the other probe is modified in the 5 '-terminus with an acceptor fluorophore. When they are in proximity fluorescence energy is transferred from the donor to the acceptor fluorophore, which can be detected.
  • the fluorophores are far apart in solution, but are brought together when the probes hybridize to TS by binding with the 3 '- termius of one probe next to the 5'-terminus of the other probe.
  • the strategy has several disadvantages. It is necessary to distinguish fluorescence intensity of different wavelengths, since hybridization does not give rise to a significant change ' in total fluorescence, but only a change in the wavelength of fluorescence.
  • the system is not suitable for quantitative determination of TS, since energy transfer efficiency depends on factors such as the distance between the fluorophores and their relative orientation (F ⁇ rster, Ann. Phys. (Leipzig) 2:55-75, 1948), which may depend on the probed sequence.
  • the strategy has fundamental problems with background fluorescence, since the light used to excite the donor does also to some degree excite the acceptor leading to a non-specific background signal. Finally, the requirement that two probes bind simultaneously to the target sequence results in slow hybridization kinetics making the technique less suitable for real time detection.
  • oligonucleotides are complementary to each other and also to the two strands of the target sequence. Both have a fluorophore in the 3 '-terminus and a quencher in the 5 '-terminus. When these pair with each other the quenchers at the 5 '-terminus are in immediate proximity of the fluorophores at the 3 '-terminus quenching their fluorescence. However, if the probe instead binds to TS fluorescence is observed.
  • Tyagi S., (PCT- WO 9513399; Nature biotech. 14, 303-307, 1989) describes 'molecular beacons' that are based on a probe with two chromophores, one at each end. These are chosen such that one chromophore quenches the fluorescence of the other when they are in proximity.
  • the probe is designed to form secondary structure in solution that brings the two ends of the probe together, resulting in fluorescence quenching.
  • This structural requirement is the first limitation of the probe since it must contain sequences that produces a particular secondary structure.
  • the probes are complementary also to other sequences than those they are designed to recognize, i.e., a probe is never unique for single TS.
  • a further disadvantage is that probing is limited to a narrow temperature range, since both the hybrid between probe and TS and the secondary structure in the free probe must be stable. Temperatures at which TS does not hybridize to complementary NA, for example, can not be used. Further, thermal motion, which is significant already at room temperature, decreases the quenching efficiency, making it often necessary to use even lower probing temperatures, which decreases the specificity of the probing reaction.
  • One objective of the present invention is to overcome the limitations discussed above with traditional methods and also the limitations of the present homogeneous methods.
  • pretreatment of the sample should not be necessary, that target sequences are detected through hybridization with a probe that generates a signal, but which in non-hybridized state generates a much smaller, preferably negligible signal, that probing is possible in a homogeneous solution, that hybridization can be demonstrated rapidly, without delay, that the amount of NA can be quantified in real time, that particular NA sequences can be demonstrated in samples containing active enzymes, such as nucleases and proteases, that presence of a particular NA can be demonstrated in vivo, that the presence of a particular NA can be demonstrated with inexpensive equipment, that presence of an arbitrary sequence can be demonstrated selectively, that probing can be performed in a large temperature range, that people using the invention should not get exposed to hazardous chemicals, and that people using the probe should not require special training or particular experience.
  • the probe generates a signal that can be detected by the naked eye.
  • the present invention fulfills these requirements to a reasonable degree.
  • the present invention is a probe composed of a sequence recognizing element (SRE) and a reporter group (RG) joint by a linker.
  • RG is a compound characterized by having an observable property altered upon binding to nucleic acids (NAs). For example it may have minimal luminescence free in solution and obtain strong luminescence when bound to NAs.
  • NAs nucleic acids
  • the invention relates to new asymmetric cyanine dyes to provide fluorescence. In a specific embodiment thereof it clearly differs between PNA and DNA, whereby it provide a strong fluorescence when attached to a DNA.
  • the here-invented probe may recognize TS in single-stranded (ss) NA as well as in double- stranded (ds) NA.
  • the here-invented probe may form complexes with ssNA that are more stable than dsNA, and may be used to probe dsNA at a temperature where its strands are separated.
  • the here-invented probe has a potential to be used for probing in vivo, both in cultivated cells and in whole organisms, where it has the advantage to traditional NA-based probes of being resistant to enzymes.
  • the here-invented probe gives rise to a signal that is proportional to the amount of TS and can be used to quantify the amount of a particular NA in a sample. This can be used to determine, for example, the amount of a particular PCR product in a complex mixture. It may also be possible to determine the amount of a particular RNA in, for example, cell extracts, or the relative concentration of two genes. The latter can, for example, be used to follow the progression of cancers.
  • the present invention generates a signal immediately upon hybridization and can be used to determine the amount of a particular NA in real time. This makes it possible to follow, for example, PCR reactions, in vitro transcription, etc., in real time. It should also be possible to monitor changes in the amount of a particular NA in cells in real time, for example, to follow the replication of chromosome or plasmid, or the production of a particular RNA.
  • Probes can be constructed that hybridize more efficiently to a fully complementary sequence than to sequences that differ in one or a few bases.
  • the present invention has presupposition to be used to localize particular sequences in chromosomes by 'fluorescence in situ hybridization' (FISH) technique. Also here the present invention has advantages to traditional FISH-probes by its increase in fluorescence upon hybridization. To obtain sufficient signal intensity it may be necessary to hybridize several probes to the target sequence and/or equip them with many RG:s.
  • FISH fluorescence in situ hybridization'
  • the present invention can be used, for example, to identify infectious agents (viruses, bacteria, parasites etc) in patients by detecting sequences specific for the foreign organisms, to test if individuals are predisposed, or suffer increased risk, to develop a disease by testing their genetic material, for prenatal diagnosis, to find genetic defects in embryos and fosters, and to predict complications in connection with, for example, birth delivery, to identify individuals in, for example, paternity tests, forensic tests, etc., to test the outcome in gene technological experiments, such as cloning, transfections,
  • the present invention is a probe for detecting and quantifying nucleic acids (NAs) containing a particular target sequence (TS).
  • NAs nucleic acids
  • TS target sequence
  • the present invention can be used for homogeneous probing; i.e., presence of TS can be demonstrated without removal of non-hybridized probe.
  • the present invention has at least one of the following advantages: higher sensitivity, higher accuracy, and faster detection.
  • the present invention is a probe composed of a sequence recognizing element (SRE) and a reporter group (RG) that are linked, the RG group consisting of a cyanine dye having the following structural formulae:
  • Xi is selected from the group consisting of O, S, Se, NR 5 , wherein R 5 is selected from the group consisting of hydrogen or an alkyl group having at most 6 carbon atoms, or , independently from each other, are hydrogen or an alkyl group having at most 6 carbon atoms, wherein X 2 represents no substitution at all, or is selected from the group consisting of hydrogen, substituted or, unsubstituted alkyl or a metallo group, such as Hg, Zn, Mg, Pd, in cooperation with an anioh such as acetate, halogenide, trifluoroacetate, wherein Ar represents an optional 5- or 6-membered aromatic group that may contain 0-2 hetero atoms such as O, S, or NR 5 , wherein R 5 has the meaning as given above, where n is 0, 1 or 2, wherein R 1 is selected from the group consisting of substituted or unsubstituted alkyl having 1 to 4 carbon atoms, in the alkyl moiety,
  • Homo- and heterodimers of the dyes can also be used in the present invention.
  • Another route of preparing these compounds is to make use of the difference in basicity of the backbone of DNA, phosphate groups, and PNA, amide groups.
  • a route for preparing neutral cyanine dyes is:
  • This dye can be in three different forms depending on pH and these dyes all show different photophysical properties compared to the pyridine (BO) derivative as such.
  • BO-neutral shows low fluorescence
  • BO-cation shows strong fluorescence
  • BO-dication shows no fluorescence when bound to nucleic acids.
  • pH 7 where the BO-cation is present the dye shows almost the same fluorescence increase upon addition to DNA or PNA.
  • pH 10 where BO-neutral is present the dye shows a weak fluorescence upon addition of PNA but shows high fluorescence upon addition of DNA. It seems that the backbone of DNA or the water near the backbone provides for protonation of BO-neutral.
  • the BO-neutral can easily be transformed to BO by reacting with alkyl halides. This way opens up to a route to make lots of BO-derivatives comprising different substituents.
  • m is an integer from 1 to 6, preferably 3 to 4
  • o is an integer from 1 to 6, preferably 2 to 4
  • R8 to Rl 1 is hydrogen, or alkyl having 1 to 6 carbon atoms, preferably methyl.
  • the following compounds of the invention have been synthetized.
  • RG is a molecule that has affinity for NA:s (it must, otherwise it would not bind to TS upon hybridization), it will have a large tendency to fold back interacting with the SRE in any NA-based probe unless precautions are taken! This is a particularly important problem in probes where RG is a cation, as in fact used in all previous probes of this kind (EPO 710668; US 5157032; Ishiguro et al., Nucl. Acids Res. 24, 4992, 1996), since the RG is attracted to the NA electrostatically.
  • NA-RG probes no matter their signal property. It may be the interaction with electromagnetic radiation, as measured through changes in absorption or luminescence (fluorescence, phosphorescence), in steady-state or time resolved fashion, or a change in NMR response, redox potential, conductivity, reactivity etc.
  • probes based on NA:s with unoptimized sequences will suffer from back binding of the RG, and have undesired background signal.
  • the present invention describes SRE-RG probes, where the problem of RG folding back onto the SRE giving rise to background signal, is minimized. It also describes how the sequence of the probe, and the probing strategy, can be optimized to improve the change in the observable property of the probe. Finally, one form of SRE-RG probes is described that also exhibits a stronger signal upon binding to TS than corresponding NA-RG probes.
  • RG is a molecule that delivers a readily detectable signal when, linked to SRE, binds TS. This signal should be significantly larger than any signal from RG linked to SRE in absence of TS. Since the number of structurally different NAA:s is very large, there is a good chance to find one that does not interact, or interacts in a way that does not give rise to the same change in the observable property of RG as natural NA:s do. Consequently, all compounds whose observable properties are altered upon binding to NA can be used as RG in combination with an appropriate SRE in a probe according to the present invention.
  • the invention is illustrated by RG:s whose spectroscopic properties are altered upon binding to NAs, which is measured as a change in total fluorescence intensity. In the examples the fluorescence signal increases upon probe binding. This is, of course, not a limitation of the present invention. Probes with RG:s whose signal property decreases upon binding to TS can also be used.
  • RG Since luminescence can be detected with very high sensitivity, compounds that obtain an increase in fluorescence upon binding to NA:s are suitable as RG. Their quantum yield of luminescence should increase at least 10-fold upon binding NA, preferable at least 50-fold and more preferably at least 500-fold. Many such compounds are known. One of those with largest increase is thiazole orange (over 5000-fold, Rye et al., Nucl. Acids Res., 11, 2803, 1992).
  • the compounds should free in solution, i.e., in absence of NAs, have very low luminescence, since this gives rise to a background signal.
  • the quantum yield of luminescence of the free compound should be less than 0.05, preferably less than 0.01 and more preferable less than 0.001.
  • the compounds should absorb light efficiently in the UV/VIS region. Its molar absorptivity at absorption maximum should be at least 1000 M-lcm "1 , preferably at least 10,000 M ⁇ cm "1 , and more preferably at least 50,000 M ⁇ cm "1 .
  • R 1 is a hydrogen or to the nitrogen non-conjugated alkyl group of at most 6 carbon atoms, that may be substituted with polar residues such as hydroxyl groups, alkoxy groups, carboxyl groups and amino groups.
  • X is O, S (or Se), N-R 5 , where R 5 is hydrogen or a small alkyl group, or CR 6 R 7 , where R 6 and R 7 are hydrogens or alkyl groups.
  • the first ring system is in these cases a benzoxazole, benzothiazole, benzimidazole and indoline, respectively.
  • the other aromatic ring system may be a single or double aromatic ring, usually a quinoline or a pyridine.
  • the side groups R 2 , R 3 and R 4 which may be same, are hydrogen, small alkyl groups, aryles, or in pair, R 2 and R 4 or R 3 and R 4 , and, in combination with two of the ring atoms, constitute a 5 or 6-membered aromatic ring, that may contain 0-2 heteroatoms such as O, S and N-R 8 , where R 8 is an alkyl group.
  • n is 0, 1 or 2. This affects the distance and degree of conjugation between the ring systems and hence the wavelengths of absorption and emission (Griffith in 'Colour and constitution of organic molecules', Academic press, 1976).
  • the affinities and luminescence properties of the asymmetric cyanine dyes depend on the base-sequence of the NA they interact with. They bind with very high affinity to dsDNA, most likely by intercalation (Jacobsen et al, Nucl. Acids Res. 23, 753, 1995; Hansen et al, Nucl Acids Res., 24, 859, 1996). They bind somewhat weaker ( ⁇ 10 fold) to single- stranded polypurines, and considerably weaker (-100 fold) to single-stranded polypyrimidines.
  • the fluorescence properties of the dyes depend on base sequence. The fluorescence is about 10 times more intense when they are bound to dsDNA and to polypurines than when they are bound to polypyrimidines. Differences are also seen in the absorption properties of the bound dye. Clearly the properties of the asymmetric cyanine dyes bound to NA:s depend very much on the base or bases with which they are interacting.
  • a probe comprising the present invention has a SRE element with a structure (either chemical or sequence or both) that either minimizes the interaction with RG, or interacts with RG such that it obtains minimal change in its signal properties.
  • the SRE is either a NA, with a particular base or bases at the end to which RG is attached, or the SRE is chemically and/or structurally different from NA:s. It may be a nucleic acid analogue (NAA) (here equivalent to an oligonucleotide analogue) that is different from natural NA:s, but recognizes them through specific pairing between nucleotide bases.
  • NAA nucleic acid analogue
  • It may also be a peptide that binds sequence specifically to NA:s, and it may be a combination of a peptide and a NAA, and, in an appropriate design, it may be a combination of a peptide and a NA. It may also be an organic synthetic molecule that binds to NA:s sequence specifically.
  • the SRE is a NAA.
  • NAA we refer to a linear polymer composed of units containing nucleotide bases, but differs from natural NA by having the phosphodiester backbone modified or replaced, or the sugar moieties modified or replaced, or has a different stereo chemistry, but interacts sequence specifically with NA through base-pair formation.
  • the NAA must be sufficiently different from NA to it interact substantially differently with RG.
  • NA derivatives i.e., NA:s with one or several hydrogen atoms substituted by other groups
  • those that can be synthesized by commercial oligonucleotide synthesizers today are not expected to be sufficiently dissimilar.
  • SRE and RG must have suitable reactive groups. Many combinations are possible.
  • Thiols such as in cystein, can be joined to other thiols and to alkylating groups, such as iodoacetamide, various maleimides, derivatives of acrylic acid etc.
  • Aminogroups such as the amino terminal and basic aminoacid residues in peptides, and in PNA, can be reacted with isothiocyanates, imidesters, such as succinimidesters and phthalimidesters, and sulfonhalides, glyoxals, aldehydes and ketones.
  • Carboxylic acids such as the carboxyl terminal in peptides and acidic amino acid residues, can be reacted with amines, hydrazin derivatives etc. These coupling reactions are well known in the art, and are described in, for example, the Novabiochem 97/98 Catalogue & Peptide synthesis handbook and the Handbook of Fluorescent Probes and Research Chemicals (sixth edition, Molecular Probes inc., ed. Richard Haugland).
  • SRE and RG conjugation of SRE and RG is illustrated by two quite different approaches; one based on solution chemistry and one based on solid phase chemistry.
  • Solid phase conjugation of RG to SRE is exemplified by attaching novel carboxylic acid derivatives of the asymmetric cyanine dyes that we have developed, to SRE:s with amino groups.
  • the solid phase approach is particularly interesting for SRE:s of the kind peptides and PNA, since the dyes may be attached by the same procedure as the aminoacid residues and the PNA-bases, and complete probes can be synthesized using commercial peptide synthesizers.
  • the linker The main function of the linker is to keep the units together in a way that does not obstruct the interaction between RG and TS upon hybridization.
  • the linker may be uncomplicated, such as a chemical single bond, but may also be complex containing, for example, functional groups. It may also be designed to obstruct the interaction between SRE anu RG. Our results, based on modeling studies, show this may be accomplished by using short and/or stiff linkers, and linkers containing bulky groups. If RG is charged, like charges may be introduced into the linker to suppress back binding.
  • Many linkers can be constructed by joining SRE with suitable derivatives of RG. More complex ones can be constructed by attaching additional units to either the SRE or the RG before joining them together.
  • Probing strategies and designs ssNA can be probed using a probe with an SRE that binds ssTS.
  • the SRE is usually a NAA , but can also be a peptide, a peptide-NAA conjugate, a NA-NAA mixed polymer, or a designed NA-peptide conjugate.
  • the size of TS may vary, depending on the system being probed. For example, quantifying the amount of a particular NA in a sample containing no, or only a few, other NA:s (i.e., such as the product of a PCR reaction), the probe may be as short as 5-6-bases, provided it forms stable hybrids and has sufficient sequence specificity.
  • NAA or NA-based probes one should chose a TS that at the end closest to RG in hybridized state has the base or bases complementary to those RG has least affinity for and/or in which presence RG obtains least signal.
  • TS should end by ..AA or ..GA. It is also an advantage if TS is close to a site, or with the probe creates a site, that is in reach for RG in the hybridized state, for which RG has high affinity and when bound to it, obtains intense signal. Similar strategy can by used to optimize the signal response of a probe that is sensitive to a particular mutation.
  • dsNA can be probed at conditions, such as for example high temperature and low ionic strength, where its strands are separated, using a probe with an SRE that forms more stable hybrids with NA than a complementary NA.
  • SRE:s are, for example, many uncharged and cationic NAA:s.
  • Native dsNA can be probed using a probe with a NAA that forms sequence specific triplexes or sequence specific D-loops as SRE, or a protein or peptide that binds dsNA as SRE, or a protein peptide-NAA/NA conjugate.
  • ssNA can also be probed by simultaneous hybridization of a probe according to this invention (based on NAA, peptide/protein, peptide-NAA conjugate or peptide-conjugate) and an oligonucleotide that are complementary to close lying regions of a TS, such that the oligonucleotide forms a duplex to which RG can bind.
  • a probe according to this invention based on NAA, peptide/protein, peptide-NAA conjugate or peptide-conjugate
  • an oligonucleotide that are complementary to close lying regions of a TS, such that the oligonucleotide forms a duplex to which RG can bind.
  • dsDNA can also be probed using two complementary probes that recognizes the two strands of TS. This approach counteracts renaturation and may produce larger signal response.
  • Probing can also be made with the probe immobilized to a solid support, preferable by a tether to SRE at the opposite end of RG. Such an approach could readily be automated, and the immobilized probes may even be reused.
  • Probing can also be made with the sample NA immobilized, as in many conventional approaches.
  • the invented probe has the advantage that the washing step to remove non- hybridized probe is less critical.
  • Probes may be constructed with two RG:s, that may be different, and whose combined observable properties are altered upon hybridization.
  • the probes can be designed as the NA-based probes with two pyrenes, as described by Yamana (Nucl. Acids Res. 11 (2-4), 383, 1992), or a fluorophore and quencher that quenches the fluorescence of the free probe by either intermolecular (Morrison, EPA 87300195.2) or intramolecular (Tyagi, WO 9513399) interactions.
  • the invented probes have the advantage of forming more stable complexes (allowing probing at temperatures above the melting temperature of dsNA) and being resistant to nucleases.
  • one RG is a fluorophore that obtains a large increase in fluorescence upon binding to NA:s, such as the asymmetric cyanine dyes, and the other is a quencher.
  • the quencher would quench any residual fluorescence of the free probe, further improving the fluorescence enhancement upon hybridization.
  • Probing can also be performed in the presence of a third component that reduces the residual fluorescence of the free probe.
  • the third component may be a quencher, i.e., a molecule that quenches the fluorescence of the RG in the free probe.
  • the quencher could be free in solution, it could also be attached to the SRE, as described above, or it can be attached to another NA or NAA that is complementary to a part of SRE.
  • This NA/NAA- quencher shall bind to the free probe in a way that brings the quencher into proximity of the RG. If the complementarity is only partial, the probe will have higher affinity for TS, and will dissociate from the NA/NAA-quencher if TS is present.
  • the third component may also be an agent that binds RG and sequesters it from the back bound position in SRE.
  • Different agents are likely to work best with different RG:s.
  • Calixarene for example, has large affinity for TO, and can be used to attenuate the background luminescence of SRE- TO probes.
  • Probes of the present invention can be used to simultaneously detect and quantify the presence of several different NA:s in a sample, by constructing them with RG:s that have distinguishable spectral responses. They may, for example, emit light of different wavelengths.
  • the present invention may also be used localize sequences in chromosomes by Fluorescence in situ hybridization (FISH).
  • FISH Fluorescence in situ hybridization
  • the invented probes have the advantage to conventional probes that background signal from non-hybridized probes is considerably lower.
  • Binding of the probe to TS can be monitored by any observable property of the probe that is altered upon binding. Since fluorescence intensity can be measured with very high sensitivity by relatively inexpensive equipment, fluorescence detection is usually the method of choice. However, also changes in other observable properties can be monitored. Changes in fluorescence lifetime and fluorescence polarization can also be measured, as well as changes in absorption, nmr response, conductivity etc.
  • the fluorescence of both free and hybridized probe decreases with temperature.
  • the former is probably due to reduced degree of residual back binding at higher temperature, while the latter is due to increased thermal fluctuations of the bases in the NA duplex, which allows for internal flexibility in RG and hence lower fluorescence.
  • the enhancement in fluorescence upon hybridization is the ratio between these signals, and is maximal at about 62°C.
  • the fluorescence of the hybridized probe drops to background level at about 75°C, which is below the melting temperature of the PNA:NA duplex, but at a temperature where essentially no oligomer is bound. This supports the hypothesis that the dye in the hybridized state is bound to the NA:NA duplex region.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention is a probe for detecting nucleic acids having a particular sequence. It is composed of two joint units. One unit is chemically different from natural nucleic acids, but has the ability to recognize a particular sequence of bases or base pairs in single or double-stranded DNA of RNA. The other unit is a compound whose detectable properties are altered upon binding to nucleic acids.

Description

TITLE
PROBE FOR ANALYSIS OF NUCLEIC ACIDS
DESCRIPTION Technical field
The invention belongs to the category probes for hybridization to nucleic acids, and in particular to fluorescence dyes used in such probes.
Such probes are used in methods where specific genes, gene segments, RNA molecules and other nucleic acids are identified. These methods are primarily used clinically, for example to test tissue, blood and urine samples, in food technology, agriculture and in biological research.
It is one object of the present invention to obtain fluorescent dyes which exhibit stronger fluorescent reactions than hitherto known ones.
A further object is to obtain fluorescent dyes that differs between DNA and PNA when attached to a probe.
Background of the invention
The development of genetically modified products and the characterization of genes in human and other mammalian diseases require reliable detection of small amounts of DNA. By having a probe consisting of PNA and a cyanine dye it is possible to detect the presence of and/or quantify a specific DNA sequence by measuring the fluorescence increase from the dye. In order to obtain more sensitive probes the binding affinity of the dyes to PNA, which results in a background fluorescence, has to be reduced.
Within hospital care as well as within food industry systems are developed for an automatic analysis of the control of bacterial and virus concentrations. Using this new technology it is hoped that it is able to provide an analysis answer on the same day as tested, i.e. more or less in real time.
One deficit belonging to the probes of toady is that they, to a certain degree are self- fluorescent, even when they do not bind to the DNA, so called background fluorescence, which results in a bad sensitivity. By developing fluorescent dyes which bind to DNA only and/or only bind to the complex that DNA and nucleic acid of the probe forms it is possible to provide a more sensitive probe.
Probes for hybridization to nucleic acids (NA), with which we refer to both deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), are used to demonstrate the presence of specific target sequences (TS) in complex mixtures. Traditional hybridization methods, as first described by Gillespie and Spiegelman (J. Mol. Biol. 12, 829, 1956), employ a probe based on an oligodeoxyribonucleotide equipped with a reporter group (RG) that usually is a radioisotope, and encompasses usually the following steps: the nucleic acid to be tested is immobilized on a paper, glass bead or plastic surface; an excess of probe complementary to the target sequence is added; the probe is allowed to hybridize; non- hybridized probe is removed; remaining probe bound to the immobilized target sequence is detected.
Non-hybridized probe is removed by extensive washing. This is usually the most time consuming and critical step in the procedure. Since the properties of non-hybridized and hybridized probe are not distinguishable, it is necessary that essentially all non-hybridized probe is removed. Since the hybridized probe is only attached through its interaction with the target sequence also some of it will be removed by washing, as well as some hybrids between TS and probe where TS was not sufficiently immobilized. Further, some probe may stick directly to the surface giving rise to a background signal. Finally, the requirement that non-hybridized probe must be removed makes in vivo and real time detection impossible.
A few methods to demonstrate hybridization without having to remove non-hybridized probe, so called homogeneous probing techniques, have been described.
Bannwarth et al., (Helvetica Chimica Acta, 71, 2085, 1988) have developed a method with probes composed of an oligodeoxyribonucleotide equipped with a ruthenium complex, where hybridization can be demonstrated from measurements of the probe fluorescence lifetime. Although the strategy is elegant, its application is limited to specialized laboratories that have sophisticated instrumentation, and can only be used by people with special training. Further, the ability of the method to distinguish hybridized and non- hybridized probe is not too good, particularly not in biological samples that may contain components that affect the probe fluorescence life time.
Barton J., (US Pat. 5.157.032) describes a probe composed of a DNA-chain modified with a metal-ligand complex whose fluorescence intensity increases upon hybridization. These probes obtain only a modest fluorescence upon hybridization (a fluorescence quantum yield of 0.007 has been reported, Jenkins & Barton, J. Am. Chem. Soc, 114, 8736, 1992), which gives low sensitivity. Further, the probes are dicationic (charge +2), which leads to considerable non-specific contribution to the interaction and consequently a decreased ability to distinguish different sequences.
Yamana et al., (Nucl. & Nucl. 11 (2-4), 383, 1992) describe a probe composed of an oligonucleotide modified with pyrene, which under optimal conditions gives a 20-fold increase in fluorescence upon hybridization. The method has several disadvantages. Pyrene has complicated photophysics and its absorption and fluorescence properties depend on its closest surrounding; for example, it has a large tendency to form excimers (J. Michl & Erik W. Thulstrup in Spectroscopy with polarized light, 1st Ed. VCH, 1986, ISBN 0-89573-346- 3). Further, pyrene emits ultraviolet light (below 450 am) that cannot be seen by the naked eye. Finally, pyrene is toxic (Yoshikawa et al, Vet. Hum. Toxicol. 29, 25, 1987).
Linn et al., (EP 0710 668 A2, US 5597696) and Ishiguro et al., (Nucl. Acids Res. 24, 4992, 1996) describes probes composed of an oligonucleotide and an asymmetric cyanine dye. The fluorescence properties, such as fluorescence polarization, fluorescence lifetime and fluorescence intensity, of these probes are changed upon hybridization. These probes have several disadvantages and limitations. Measurements of fluorescence polarization and fluorescence lifetime require sophisticated and expensive instrumentation, and must be performed by people with specialist training. The change in fluorescence intensity is modest (a 4-fold increase under optimal conditions has been reported), making probing very sensitive to background, particularly at conditions that require excess of probe.
Heller et al, (EPA 070685) and Cardullo et al., (Proc. Natl. Acad. Sci. USA, 85, 8790-8794, 1988) describe a probe based on simultaneous hybridization of two DNA-based probes to close-lying sequences. One probe is modified in the 3 '-terminus of the DNA chain with a donor fluorophore and the other probe is modified in the 5 '-terminus with an acceptor fluorophore. When they are in proximity fluorescence energy is transferred from the donor to the acceptor fluorophore, which can be detected. The fluorophores are far apart in solution, but are brought together when the probes hybridize to TS by binding with the 3 '- termius of one probe next to the 5'-terminus of the other probe. The strategy has several disadvantages. It is necessary to distinguish fluorescence intensity of different wavelengths, since hybridization does not give rise to a significant change' in total fluorescence, but only a change in the wavelength of fluorescence. The system is not suitable for quantitative determination of TS, since energy transfer efficiency depends on factors such as the distance between the fluorophores and their relative orientation (Fδrster, Ann. Phys. (Leipzig) 2:55-75, 1948), which may depend on the probed sequence. The strategy has fundamental problems with background fluorescence, since the light used to excite the donor does also to some degree excite the acceptor leading to a non-specific background signal. Finally, the requirement that two probes bind simultaneously to the target sequence results in slow hybridization kinetics making the technique less suitable for real time detection.
Another technique based on a pair of oligonucleotides was described by Morrison (EPA 87300195.2; US. Pat. 4822733; Analyt. Biochem. 183, 231-244, 1989; Biochem. 32, 3095- 3104, 1993). These oligonucleotides are complementary to each other and also to the two strands of the target sequence. Both have a fluorophore in the 3 '-terminus and a quencher in the 5 '-terminus. When these pair with each other the quenchers at the 5 '-terminus are in immediate proximity of the fluorophores at the 3 '-terminus quenching their fluorescence. However, if the probe instead binds to TS fluorescence is observed. With this strategy one has two opposing design problems: It is desirable to have a high probe concentration to obtain fast hybridization kinetics, but simultaneously it is desirable to have a low probe concentration to minimize the background luminescence from free probes that have found neither TS nor a complementary probe to bind. Probing is performed by first heating the sample to separate the strands of both the probe molecules and the dsNA, and then the temperature is lowered to allow the probe to hybridize to TS. Unhybridized probe must, however, find a complementary probe to become quenched, until then it give rise to the same signal as probes hybridized to TS. Since probe is usually used in large excess, it make take considerable time before the background has dropped to an acceptable level making the strategy unsuitable for real-time detection. Finally, these probes are only applicable to double stranded TS.
Tyagi, S., (PCT- WO 9513399; Nature biotech. 14, 303-307, 1989) describes 'molecular beacons' that are based on a probe with two chromophores, one at each end. These are chosen such that one chromophore quenches the fluorescence of the other when they are in proximity. The probe is designed to form secondary structure in solution that brings the two ends of the probe together, resulting in fluorescence quenching. This structural requirement is the first limitation of the probe since it must contain sequences that produces a particular secondary structure. As a consequence the probes are complementary also to other sequences than those they are designed to recognize, i.e., a probe is never unique for single TS. A further disadvantage is that probing is limited to a narrow temperature range, since both the hybrid between probe and TS and the secondary structure in the free probe must be stable. Temperatures at which TS does not hybridize to complementary NA, for example, can not be used. Further, thermal motion, which is significant already at room temperature, decreases the quenching efficiency, making it often necessary to use even lower probing temperatures, which decreases the specificity of the probing reaction. One objective of the present invention is to overcome the limitations discussed above with traditional methods and also the limitations of the present homogeneous methods.
Further objectives with the present invention are: that pretreatment of the sample, such as degradation to smalle fragments, should not be necessary, that target sequences are detected through hybridization with a probe that generates a signal, but which in non-hybridized state generates a much smaller, preferably negligible signal, that probing is possible in a homogeneous solution, that hybridization can be demonstrated rapidly, without delay, that the amount of NA can be quantified in real time, that particular NA sequences can be demonstrated in samples containing active enzymes, such as nucleases and proteases, that presence of a particular NA can be demonstrated in vivo, that the presence of a particular NA can be demonstrated with inexpensive equipment, that presence of an arbitrary sequence can be demonstrated selectively, that probing can be performed in a large temperature range, that people using the invention should not get exposed to hazardous chemicals, and that people using the probe should not require special training or particular experience.
To be able to utilize the entire potential of hybridization methods in diagnosis and research it is necessary to have a technique to detect hybridization in a solution using probes that by themselves generate low or negligible signals, but produce an observable response upon hybridization to target sequence. It is also desirable that the probe can be used in vivo without having a deleterious effect on tissue and cells. It should also allow real time detection. Of course, it should also be possible to use the probe for traditional hybridization.
It is also desirable that the probe generates a signal that can be detected by the naked eye.
The present invention fulfills these requirements to a reasonable degree.
Summary of the present invention
The present invention is a probe composed of a sequence recognizing element (SRE) and a reporter group (RG) joint by a linker. RG is a compound characterized by having an observable property altered upon binding to nucleic acids (NAs). For example it may have minimal luminescence free in solution and obtain strong luminescence when bound to NAs. Of crucial importance is that the RG and SRE, when joint, interact with each other substantially differently than RG interacts with the target upon hybridization. In particular the invention relates to new asymmetric cyanine dyes to provide fluorescence. In a specific embodiment thereof it clearly differs between PNA and DNA, whereby it provide a strong fluorescence when attached to a DNA.
The here-invented probe may recognize TS in single-stranded (ss) NA as well as in double- stranded (ds) NA.
The here-invented probe may form complexes with ssNA that are more stable than dsNA, and may be used to probe dsNA at a temperature where its strands are separated.
The here-invented probe has a potential to be used for probing in vivo, both in cultivated cells and in whole organisms, where it has the advantage to traditional NA-based probes of being resistant to enzymes.
The here-invented probe gives rise to a signal that is proportional to the amount of TS and can be used to quantify the amount of a particular NA in a sample. This can be used to determine, for example, the amount of a particular PCR product in a complex mixture. It may also be possible to determine the amount of a particular RNA in, for example, cell extracts, or the relative concentration of two genes. The latter can, for example, be used to follow the progression of cancers.
The present invention generates a signal immediately upon hybridization and can be used to determine the amount of a particular NA in real time. This makes it possible to follow, for example, PCR reactions, in vitro transcription, etc., in real time. It should also be possible to monitor changes in the amount of a particular NA in cells in real time, for example, to follow the replication of chromosome or plasmid, or the production of a particular RNA.
The present invention can also be used to detect mutations. Probes can be constructed that hybridize more efficiently to a fully complementary sequence than to sequences that differ in one or a few bases.
The present invention has presupposition to be used to localize particular sequences in chromosomes by 'fluorescence in situ hybridization' (FISH) technique. Also here the present invention has advantages to traditional FISH-probes by its increase in fluorescence upon hybridization. To obtain sufficient signal intensity it may be necessary to hybridize several probes to the target sequence and/or equip them with many RG:s.
The present invention can be used, for example, to identify infectious agents (viruses, bacteria, parasites etc) in patients by detecting sequences specific for the foreign organisms, to test if individuals are predisposed, or suffer increased risk, to develop a disease by testing their genetic material, for prenatal diagnosis, to find genetic defects in embryos and fosters, and to predict complications in connection with, for example, birth delivery, to identify individuals in, for example, paternity tests, forensic tests, etc., to test the outcome in gene technological experiments, such as cloning, transfections,
'gene-knockouts' and the like.
Detailed description of the invention and its preferred embodiments
As indicated by the title, the present invention is a probe for detecting and quantifying nucleic acids (NAs) containing a particular target sequence (TS). In contrast to traditional methods, the present invention can be used for homogeneous probing; i.e., presence of TS can be demonstrated without removal of non-hybridized probe. Compared to existing homogeneous probing methods the present invention has at least one of the following advantages: higher sensitivity, higher accuracy, and faster detection.
The present invention is a probe composed of a sequence recognizing element (SRE) and a reporter group (RG) that are linked, the RG group consisting of a cyanine dye having the following structural formulae:
Figure imgf000008_0001
wherein Xi is selected from the group consisting of O, S, Se, NR5, wherein R5 is selected from the group consisting of hydrogen or an alkyl group having at most 6 carbon atoms, or
Figure imgf000008_0002
, independently from each other, are hydrogen or an alkyl group having at most 6 carbon atoms, wherein X2 represents no substitution at all, or is selected from the group consisting of hydrogen, substituted or, unsubstituted alkyl or a metallo group, such as Hg, Zn, Mg, Pd, in cooperation with an anioh such as acetate, halogenide, trifluoroacetate, wherein Ar represents an optional 5- or 6-membered aromatic group that may contain 0-2 hetero atoms such as O, S, or NR5, wherein R5 has the meaning as given above, where n is 0, 1 or 2, wherein R1 is selected from the group consisting of substituted or unsubstituted alkyl having 1 to 4 carbon atoms, in the alkyl moiety, or substituted or unsubstituted carboxy having 1 to 10 carbon atoms in the alkyl moiety preceding the carboxy group, or an alkylamine having 1-3 carbon atoms in the alkylgroup, which alkylamino group may be further substituted by a benzthiazole group, benzimidazole group, wherein R2 is selected from the group consisting of halogen, nitro, sulfono or amino
Homo- and heterodimers of the dyes can also be used in the present invention.
These compounds can be synthesized with established methods (Sprague, US 2269234, 1942; Brooker et al., Houbenweyl methoden der organischen chemie, band V/ld, 1972; Lee et al., Cytometry 7, 508, 1986; Lee & Mize, EP 0410806, 1989). Their large enhancement in luminescence upon binding nucleic acids is well known (Lee & Chen, EP 0226272), and is exploited to detect reticulocytes (Lee & Chen, U.S. Pat. 4883867), parasites in blood (Lee & Mize, U. S. Pat. 4937198), to stain nucleic acids in electrophoresis (Quesada et al., Biotechniques 10, 616, 1991; Mathies and Huang, Nature 359, 167, 1992) and for ultra-sensitive detection of nucleic acids in capillary electrophoresis (Schwartz and Uhlfelder, Anal. Chem. 64, 1737, 1992). Substituted with polycationic chains (Glazer and Benson, US Pat. 5312921; Yue et al., US Pat. 5321130), and as homo and heterodimers (Yue and Haugland, US Pat. 5410030), these compounds are among the most common dyes for staining nucleic acids.
Another route of preparing these compounds is to make use of the difference in basicity of the backbone of DNA, phosphate groups, and PNA, amide groups. A route for preparing neutral cyanine dyes is:
Figure imgf000009_0001
This dye can be in three different forms depending on pH and these dyes all show different photophysical properties compared to the pyridine (BO) derivative as such. BO-neutral shows low fluorescence, BO-cation shows strong fluorescence, and BO-dication shows no fluorescence when bound to nucleic acids. At pH 7 where the BO-cation is present the dye shows almost the same fluorescence increase upon addition to DNA or PNA. But at pH 10, where BO-neutral is present the dye shows a weak fluorescence upon addition of PNA but shows high fluorescence upon addition of DNA. It seems that the backbone of DNA or the water near the backbone provides for protonation of BO-neutral.
The BO-neutral can easily be transformed to BO by reacting with alkyl halides. This way opens up to a route to make lots of BO-derivatives comprising different substituents.
The neutral dyes are tethered via the benzothiazole nitrogen to a probe in accordance with the formula below showing one embodiment in which B stands for nucleic acids
Figure imgf000010_0001
Another group of compounds are those of the general formula
Figure imgf000010_0002
wherein m is an integer from 1 to 6, preferably 3 to 4, o is an integer from 1 to 6, preferably 2 to 4, and
R8 to Rl 1 is hydrogen, or alkyl having 1 to 6 carbon atoms, preferably methyl. The following compounds of the invention have been synthetized.
Figure imgf000011_0001
It has turned out that these fluorescent dyes possess a higher photostability than previously known ones, as well as the neutral ones provide a possibility of distinguishing between PNA and DNA, as when bound to PNA at a high pH they will provide very small fluorescence.
Synthesis l l-Benzothiazolium-3-yl-undecanoic acid bromide (A)
2.6 g of 11 -Br-undecanoic acid and 1.3 g of benzothiazole were heated (110° C) for 6 hours. Acetone was carefully added and the reaction was stirred for 30 minutes. The precipitate was collected. Yield 3.0 g.
l l-(2-Pyridin-4-yl-methylene-benzothiazol-3-yl)-undecanoic acid (B)
To 0.4 g of and 0.1 g of picoline in 5 ml of methyl enechloride acetic acid anhydride was added. The reaction was stirred at room temperature over night. The mixture was filtered and the organic phase was evaporated. The product was purified by chromatography. Yield 0.1 g. 1 l-(2-Thiomethyl-benzothiazolium-3-yl)-undecanoic acid bromide (C) 2-Methylthiobenzothiazole (30 g, 0.2 mole) in toluene ( 30 ml) was heated to 80° C. 11- bromoundecanoic acid (50 g, 0.2 mole) dissolved in toluene (30 ml) was then added and the reaction was refluxed for 24 hours. The reaction mixture was poured into acetonitrile and stirred for 1 hour. The product was collected by filtration. Yield 65 g.
TO-N'-IO-COOH (D)
Freshly distilled quinoline (13 g; 0.1 moles) dissolved in toluene and methylparatoluene sulphate (18.6 g; 0.1 moles) dissolved in toluene (10 mis) were refluxed for 4 hrs. An oil formed was allowed to stand at ambient temperature over night. No crystals precipitated. The solution was heated and was poured into ethyl acetate after four hours under vigorous stirring. A precipitation of crystals was obtained. Yield: 28 g
Figure imgf000012_0001
(A) (B)
Figure imgf000012_0002
(C) (D)
Previous probes based on the SRE-RG concept have problem with a high background signal. When RG is a fluorofore, the free probe has considerable background luminescence and the increase in fluorescence intensity upon hybridization to TS is modest (less than fourfold, EP 0710 668 A2, Ishiguro et al., Nucl. Acids Res. 24, 4992, 1996). The large background signal is primarily due to that RG folds back onto the SRE and interacts with it (Figure 2). The reason previous probes suffer this problem is that they have an SRE that is based on a NA, whose sequence structure has not been optimized to minimize the signal from RG. Since RG is a molecule that has affinity for NA:s (it must, otherwise it would not bind to TS upon hybridization), it will have a large tendency to fold back interacting with the SRE in any NA-based probe unless precautions are taken! This is a particularly important problem in probes where RG is a cation, as in fact used in all previous probes of this kind (EPO 710668; US 5157032; Ishiguro et al., Nucl. Acids Res. 24, 4992, 1996), since the RG is attracted to the NA electrostatically.
The problems above are common to all NA-RG probes, no matter their signal property. It may be the interaction with electromagnetic radiation, as measured through changes in absorption or luminescence (fluorescence, phosphorescence), in steady-state or time resolved fashion, or a change in NMR response, redox potential, conductivity, reactivity etc. In all cases, probes based on NA:s with unoptimized sequences will suffer from back binding of the RG, and have undesired background signal.
The present invention describes SRE-RG probes, where the problem of RG folding back onto the SRE giving rise to background signal, is minimized. It also describes how the sequence of the probe, and the probing strategy, can be optimized to improve the change in the observable property of the probe. Finally, one form of SRE-RG probes is described that also exhibits a stronger signal upon binding to TS than corresponding NA-RG probes.
The Reporter Group
RG is a molecule that delivers a readily detectable signal when, linked to SRE, binds TS. This signal should be significantly larger than any signal from RG linked to SRE in absence of TS. Since the number of structurally different NAA:s is very large, there is a good chance to find one that does not interact, or interacts in a way that does not give rise to the same change in the observable property of RG as natural NA:s do. Consequently, all compounds whose observable properties are altered upon binding to NA can be used as RG in combination with an appropriate SRE in a probe according to the present invention. The invention is illustrated by RG:s whose spectroscopic properties are altered upon binding to NAs, which is measured as a change in total fluorescence intensity. In the examples the fluorescence signal increases upon probe binding. This is, of course, not a limitation of the present invention. Probes with RG:s whose signal property decreases upon binding to TS can also be used.
Since luminescence can be detected with very high sensitivity, compounds that obtain an increase in fluorescence upon binding to NA:s are suitable as RG. Their quantum yield of luminescence should increase at least 10-fold upon binding NA, preferable at least 50-fold and more preferably at least 500-fold. Many such compounds are known. One of those with largest increase is thiazole orange (over 5000-fold, Rye et al., Nucl. Acids Res., 11, 2803, 1992).
The compounds should free in solution, i.e., in absence of NAs, have very low luminescence, since this gives rise to a background signal. The quantum yield of luminescence of the free compound should be less than 0.05, preferably less than 0.01 and more preferable less than 0.001.
The compounds should absorb light efficiently in the UV/VIS region. Its molar absorptivity at absorption maximum should be at least 1000 M-lcm"1, preferably at least 10,000 M^cm"1, and more preferably at least 50,000 M^cm"1.
Prior art discloses asymmetric cyanine dyes (F. M. Hamer, in Heterocyclic compounds, Vol. 18 'Cyanine dyes and related compounds', 1964, Wiley & Sons), such as those described in (US 4883867; US 5312921; US 5321130; US 5401847; US 5410030; US 5436134, US 5486616), and particularly those with the structures shown below:
Figure imgf000014_0001
where R1 is a hydrogen or to the nitrogen non-conjugated alkyl group of at most 6 carbon atoms, that may be substituted with polar residues such as hydroxyl groups, alkoxy groups, carboxyl groups and amino groups. X is O, S (or Se), N-R5, where R5 is hydrogen or a small alkyl group, or CR6R7, where R6 and R7 are hydrogens or alkyl groups. The first ring system is in these cases a benzoxazole, benzothiazole, benzimidazole and indoline, respectively. The other aromatic ring system may be a single or double aromatic ring, usually a quinoline or a pyridine. The side groups R2, R3 and R4, which may be same, are hydrogen, small alkyl groups, aryles, or in pair, R2 and R4 or R3 and R4, and, in combination with two of the ring atoms, constitute a 5 or 6-membered aromatic ring, that may contain 0-2 heteroatoms such as O, S and N-R8, where R8 is an alkyl group. In the methine bond that connects the two aromatic systems n is 0, 1 or 2. This affects the distance and degree of conjugation between the ring systems and hence the wavelengths of absorption and emission (Griffith in 'Colour and constitution of organic molecules', Academic press, 1976). Y is HC=CH and its position is given by the indexes A and B, which are 0 or 1, if A=0 then B=l, and vice versa. The affinities and luminescence properties of the asymmetric cyanine dyes depend on the base-sequence of the NA they interact with. They bind with very high affinity to dsDNA, most likely by intercalation (Jacobsen et al, Nucl. Acids Res. 23, 753, 1995; Hansen et al, Nucl Acids Res., 24, 859, 1996). They bind somewhat weaker (< 10 fold) to single- stranded polypurines, and considerably weaker (-100 fold) to single-stranded polypyrimidines. Also the fluorescence properties of the dyes depend on base sequence. The fluorescence is about 10 times more intense when they are bound to dsDNA and to polypurines than when they are bound to polypyrimidines. Differences are also seen in the absorption properties of the bound dye. Clearly the properties of the asymmetric cyanine dyes bound to NA:s depend very much on the base or bases with which they are interacting.
Molecules tethered to dsNA:s, and therefore most likely also to ssNA:s and NAA:s, interact mainly with the 1-3 outermost bases (Ciepek et al., J. Biomol. Struct. Dyn., 5, 361, 1987). Hence it is possible to minimize the background signal of NA-based and NAA- based probes by choosing the bases nearest the end to which RG is attached. These bases should be those for which RG has least affinity and/or with which RG interacts in a way that has minimal affect on its signal properties.
For the asymmetric cyanine dyes very low background luminescence is obtained when the endmost bases of the NA or NAA based SRE are ..CT— RG or TT— RG.
The Sequence Recognizing Element
A probe comprising the present invention has a SRE element with a structure (either chemical or sequence or both) that either minimizes the interaction with RG, or interacts with RG such that it obtains minimal change in its signal properties. The SRE is either a NA, with a particular base or bases at the end to which RG is attached, or the SRE is chemically and/or structurally different from NA:s. It may be a nucleic acid analogue (NAA) (here equivalent to an oligonucleotide analogue) that is different from natural NA:s, but recognizes them through specific pairing between nucleotide bases. It may also be a peptide that binds sequence specifically to NA:s, and it may be a combination of a peptide and a NAA, and, in an appropriate design, it may be a combination of a peptide and a NA. It may also be an organic synthetic molecule that binds to NA:s sequence specifically.
In the preferred embodiment the SRE is a NAA. With a NAA we refer to a linear polymer composed of units containing nucleotide bases, but differs from natural NA by having the phosphodiester backbone modified or replaced, or the sugar moieties modified or replaced, or has a different stereo chemistry, but interacts sequence specifically with NA through base-pair formation. The NAA must be sufficiently different from NA to it interact substantially differently with RG. Hence, NA derivatives (i.e., NA:s with one or several hydrogen atoms substituted by other groups), such as those that can be synthesized by commercial oligonucleotide synthesizers today, are not expected to be sufficiently dissimilar.
Conjugation of SRE and RG
To be joined SRE and RG must have suitable reactive groups. Many combinations are possible. Thiols, such as in cystein, can be joined to other thiols and to alkylating groups, such as iodoacetamide, various maleimides, derivatives of acrylic acid etc. Aminogroups, such as the amino terminal and basic aminoacid residues in peptides, and in PNA, can be reacted with isothiocyanates, imidesters, such as succinimidesters and phthalimidesters, and sulfonhalides, glyoxals, aldehydes and ketones. Carboxylic acids, such as the carboxyl terminal in peptides and acidic amino acid residues, can be reacted with amines, hydrazin derivatives etc. These coupling reactions are well known in the art, and are described in, for example, the Novabiochem 97/98 Catalogue & Peptide synthesis handbook and the Handbook of Fluorescent Probes and Research Chemicals (sixth edition, Molecular Probes inc., ed. Richard Haugland).
The conjugation of SRE and RG is illustrated by two quite different approaches; one based on solution chemistry and one based on solid phase chemistry.
Solid phase conjugation of RG to SRE is exemplified by attaching novel carboxylic acid derivatives of the asymmetric cyanine dyes that we have developed, to SRE:s with amino groups. The solid phase approach is particularly interesting for SRE:s of the kind peptides and PNA, since the dyes may be attached by the same procedure as the aminoacid residues and the PNA-bases, and complete probes can be synthesized using commercial peptide synthesizers.
The linker
The main function of the linker is to keep the units together in a way that does not obstruct the interaction between RG and TS upon hybridization. The linker may be uncomplicated, such as a chemical single bond, but may also be complex containing, for example, functional groups. It may also be designed to obstruct the interaction between SRE anu RG. Our results, based on modeling studies, show this may be accomplished by using short and/or stiff linkers, and linkers containing bulky groups. If RG is charged, like charges may be introduced into the linker to suppress back binding. Many linkers can be constructed by joining SRE with suitable derivatives of RG. More complex ones can be constructed by attaching additional units to either the SRE or the RG before joining them together.
Probing strategies and designs ssNA can be probed using a probe with an SRE that binds ssTS. The SRE is usually a NAA , but can also be a peptide, a peptide-NAA conjugate, a NA-NAA mixed polymer, or a designed NA-peptide conjugate. The size of TS may vary, depending on the system being probed. For example, quantifying the amount of a particular NA in a sample containing no, or only a few, other NA:s (i.e., such as the product of a PCR reaction), the probe may be as short as 5-6-bases, provided it forms stable hybrids and has sufficient sequence specificity. On the other hand, when probing the presence of a particular NA in a sample containing excess of other NAs, such as the presence of foreign NA against genomic DNA, the TS has to be longer. Probes recognizing 15-40 bases will work in most cases and length of 15-25 bases are likely to be sufficient for most human samples.
When probing the presence of a long NA fragment, such as a bacterial or viral genome, or the presence of a plasmid, a particular insert etc., the probe can be designed against many segments that all may be unique to this NA. For example, a 500 bases long NA fragment has 500-20+1 = 481 segments that are 20 bases long, which all may be unique to this NA. From the point of specificity, the probe can be designed to recognize any of these segments. When using NAA or NA-based probes one should chose a TS that at the end closest to RG in hybridized state has the base or bases complementary to those RG has least affinity for and/or in which presence RG obtains least signal. For RG:s based on asymmetric cyanine dyes, such as TO and BO, TS should end by ..AA or ..GA. It is also an advantage if TS is close to a site, or with the probe creates a site, that is in reach for RG in the hybridized state, for which RG has high affinity and when bound to it, obtains intense signal. Similar strategy can by used to optimize the signal response of a probe that is sensitive to a particular mutation.
dsNA can be probed at conditions, such as for example high temperature and low ionic strength, where its strands are separated, using a probe with an SRE that forms more stable hybrids with NA than a complementary NA. Such SRE:s are, for example, many uncharged and cationic NAA:s.
Native dsNA can be probed using a probe with a NAA that forms sequence specific triplexes or sequence specific D-loops as SRE, or a protein or peptide that binds dsNA as SRE, or a protein peptide-NAA/NA conjugate.
ssNA can also be probed by simultaneous hybridization of a probe according to this invention (based on NAA, peptide/protein, peptide-NAA conjugate or peptide-conjugate) and an oligonucleotide that are complementary to close lying regions of a TS, such that the oligonucleotide forms a duplex to which RG can bind.
dsDNA can also be probed using two complementary probes that recognizes the two strands of TS. This approach counteracts renaturation and may produce larger signal response.
Probing can also be made with the probe immobilized to a solid support, preferable by a tether to SRE at the opposite end of RG. Such an approach could readily be automated, and the immobilized probes may even be reused.
Probing can also be made with the sample NA immobilized, as in many conventional approaches. Here the invented probe has the advantage that the washing step to remove non- hybridized probe is less critical.
Probes may be constructed with two RG:s, that may be different, and whose combined observable properties are altered upon hybridization. The probes can be designed as the NA-based probes with two pyrenes, as described by Yamana (Nucl. Acids Res. 11 (2-4), 383, 1992), or a fluorophore and quencher that quenches the fluorescence of the free probe by either intermolecular (Morrison, EPA 87300195.2) or intramolecular (Tyagi, WO 9513399) interactions. Here the invented probes have the advantage of forming more stable complexes (allowing probing at temperatures above the melting temperature of dsNA) and being resistant to nucleases. Of particular interest are probes where one RG is a fluorophore that obtains a large increase in fluorescence upon binding to NA:s, such as the asymmetric cyanine dyes, and the other is a quencher. In such a design the quencher would quench any residual fluorescence of the free probe, further improving the fluorescence enhancement upon hybridization.
Probing can also be performed in the presence of a third component that reduces the residual fluorescence of the free probe. The third component may be a quencher, i.e., a molecule that quenches the fluorescence of the RG in the free probe. The quencher could be free in solution, it could also be attached to the SRE, as described above, or it can be attached to another NA or NAA that is complementary to a part of SRE. This NA/NAA- quencher shall bind to the free probe in a way that brings the quencher into proximity of the RG. If the complementarity is only partial, the probe will have higher affinity for TS, and will dissociate from the NA/NAA-quencher if TS is present. The third component may also be an agent that binds RG and sequesters it from the back bound position in SRE. Different agents are likely to work best with different RG:s. Calixarene, for example, has large affinity for TO, and can be used to attenuate the background luminescence of SRE- TO probes.
Probes of the present invention can be used to simultaneously detect and quantify the presence of several different NA:s in a sample, by constructing them with RG:s that have distinguishable spectral responses. They may, for example, emit light of different wavelengths.
The present invention may also be used localize sequences in chromosomes by Fluorescence in situ hybridization (FISH). Here the invented probes have the advantage to conventional probes that background signal from non-hybridized probes is considerably lower. Of particular interest are probes equipped with several RG:s, for example, attached to branched linkers, and to other SRE components, such as the backbone, sugar moieties and nucleotide bases of NAAs.
Detection of hybridization
Binding of the probe to TS can be monitored by any observable property of the probe that is altered upon binding. Since fluorescence intensity can be measured with very high sensitivity by relatively inexpensive equipment, fluorescence detection is usually the method of choice. However, also changes in other observable properties can be monitored. Changes in fluorescence lifetime and fluorescence polarization can also be measured, as well as changes in absorption, nmr response, conductivity etc.
The fluorescence of both free and hybridized probe decreases with temperature. The former is probably due to reduced degree of residual back binding at higher temperature, while the latter is due to increased thermal fluctuations of the bases in the NA duplex, which allows for internal flexibility in RG and hence lower fluorescence. The enhancement in fluorescence upon hybridization is the ratio between these signals, and is maximal at about 62°C.
The fluorescence of the hybridized probe drops to background level at about 75°C, which is below the melting temperature of the PNA:NA duplex, but at a temperature where essentially no oligomer is bound. This supports the hypothesis that the dye in the hybridized state is bound to the NA:NA duplex region.

Claims

CLAIMS.
1. A probe for target nucleic acid sequences (TS) comprising a sequence recognizing element (SRE) covalently bound to reporter group(-s) (RG) the signal properties of which are considerably altered when it binds to a nucleic acid (NA) comprising said target sequence (TS), wherein said probe at least in its part close to the RG, has such a structure that any intramolecular interaction which affects the RG signal between the SRE and the
RG is suppressed, or has a base sequence at least at the terminal close to RG that minimally affects the said RG signal properties, characterized in that the reporter group is a compound that contains aromatic moieties out of which at least two are joined by a covalent linkage that is in conjugation with aromatic system, that the reporter group is an asymmetric cyanine compound, that the asymmetric cyanine compound has in its unbound form the following chemical structure:
Figure imgf000020_0001
wherein Xi is selected from the group consisting of O, S, Se, NR5, wherein R5 is selected from the group consisting of hydrogen or an alkyl group having at most 6 carbon atoms, or CR6R7, wherein R6 and R , independently from each other, are hydrogen or an alkyl group having at most 6 carbon atoms, wherein X2 represents no substitution at all, or is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl or a metallo group such as Hg, Zn, Mg, or Pd, wherein Ar represents an optional 5- or 6-membered aromatic group that may contain 0-2 hetero atoms such as O, S, or NR5, wherein R5 has the meaning as given above, whereby the alkylene bridge is attached in para-position to the pyridine-N, attached to the where n is 0, 1 or 2, wherein R1 is selected from the group consisting of substituted or unsubstituted alkyl having lto 4 carbon atoms, in the alkyl moiety, or substituted or unsubstituted carboxy having 1 to 10 carbon atoms in the alkyl moiety preceding the carboxy group, or an alkylamino having 1-3 carbon atoms in the alkyl group which may be further substituted by a symmetric of unsymmetric group, and wherein R is selected from the group consisting of halogen, nitro, sulfonyl, or amino
2. Probe according to claim 1, wherein X2 is no substitution at all.
3. Probe according to one or more of claims 1-2, wherein SRE and RG are linked to each other via a hydrocarbon chain containing one or more of: a) stiff groups, such as double or triple bonds, b) hetero atoms, c) polar groups, d) charged groups, and e) bulky groups.
4. Probe according to one or more of claims 1-3, wherein the linkage between SRE and RG has at least one positive charge.
5. Probe according to one or more of claims 1-4, wherein the SRE is an NAA or NA, and more than 50% of its bases are of the pyrimidine kind.
6. Probe according to one or more of claims 1-5, wherein there is a pyrimidine base in at least every second position.
7. Probe according to one or more of claims 1-6, wherein the bases at the end to which RG is attached , are two thymines, or a cytosine and a thymine.
8. Probe according to one or more of claims 1-7, wherein all bases are pyrimi dines.
9. Process of attaching a compound having one of the following chemical structures:
Figure imgf000022_0001
wherein X\ is selected from the group consisting of O, S, Se, NR , wherein R is selected from the group consisting of hydrogen or an alkyl group having at most 6 carbon atoms, or
Figure imgf000022_0002
, independently from each other, are hydrogen or an alkyl group having at most 6 carbon atoms, wherein X represents no substitution at all, or is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl or a metallo group, wherein Ar represents an optional 5- or 6-membered aromatic group that may contain 0-2 hetero atoms such as O, S, or NR5, wherein R5 has the meaning as given above, where n is 0, 1 or 2, wherein R1 is selected from the group consisting of substituted or unsubstituted alkyl having ltolO carbon atoms, in the alkyl moiety, or substituted or unsubstituted carboxy having 1 to 10 carbon atoms in the alkyl moiety preceding the carboxy group, wherein R2 is selected from the group consisting of halogen, nitro or amino
10. Process to detect a target sequence (TS) in a dsNA, without prior separation of its strands, using a probe in accordance with claims 1-9.
11. Process to detect a target sequence in a dsNA by hybridizing a probe in accordance with claims 1-9, to one of its strands at conditions where the dsNA is unstable.
12. Process to detect or quantify a particular NA in a sample containing active enzymes using a probe in accordance with claims 1-9.
13. Process to quantify the amount of a certain NA in real time, in a sample that may contain NA modifying, NA degrading, and NA synthesizing enzymes, using a probe in accordance with one or more of claims 1-9.
14. Process to detect a TS in a ssNA by hybridizing a probe in accordance with claims 1-9 and an oligomer to close-lying parts of said TS, such that RG interacts with the duplex region formed by the oligomer.
15. Process to detect or quantify NA, using a probe in accordance with one or more of claims 1-9, wherein said probe is immobilized.
16. Probe according to claims 1-9 containing more than one RG, which RGs may be identical, and whose individual or combined signal properties are altered..
17. Probe according to one or more of claims 1-9, claim 16, containing at least one RG linked to either one or more out of a) nucleotide bases b) back-bone atoms, c) sugar atoms and/or atoms composing the linker between SRE and TS.
PCT/SE2000/001429 1999-07-05 2000-07-05 Probe for analysis of nucleic acids WO2001002558A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60427/00A AU6042700A (en) 1999-07-05 2000-07-05 Probe for analysis of nucleic acids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9902565-2 1999-07-05
SE9902565A SE9902565D0 (en) 1999-07-05 1999-07-05 Probe for analysis of nucleic acids

Publications (2)

Publication Number Publication Date
WO2001002558A1 true WO2001002558A1 (en) 2001-01-11
WO2001002558A8 WO2001002558A8 (en) 2001-04-19

Family

ID=20416379

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2000/001429 WO2001002558A1 (en) 1999-07-05 2000-07-05 Probe for analysis of nucleic acids

Country Status (3)

Country Link
AU (1) AU6042700A (en)
SE (1) SE9902565D0 (en)
WO (1) WO2001002558A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073118A2 (en) * 2000-03-29 2001-10-04 Lgc (Teddington) Limited Hybridisation beacon and method of rapid sequence detection and discrimination
WO2001094473A1 (en) * 2000-06-07 2001-12-13 Lightup Technologies Ab Solid phase synthesis of cyanine dyes
WO2002014555A2 (en) * 2000-08-11 2002-02-21 University Of Utah Research Foundation Single-labeled oligonucleotide probes
EP2436780A3 (en) * 2007-10-02 2012-12-19 Charles Brush Monomethine dyes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017397A1 (en) * 1993-01-27 1994-08-04 The Regents Of The University Of California Dna complexes with dyes designed for energy transfer as fluorescent markers
EP0714986A1 (en) * 1994-12-01 1996-06-05 Tosoh Corporation Method of detecting specific nucleic acid sequences
WO1997045539A1 (en) * 1996-05-31 1997-12-04 Mikael Kubista Probe for analysis of nucleic acids
WO1999013105A1 (en) * 1997-09-05 1999-03-18 Mikael Kubista Method for the preparation of a probe for nucleic acid hybridization
WO1999037717A1 (en) * 1998-01-23 1999-07-29 The Perkin-Elmer Corporation Asymmetric cyanine dye quenchers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017397A1 (en) * 1993-01-27 1994-08-04 The Regents Of The University Of California Dna complexes with dyes designed for energy transfer as fluorescent markers
EP0714986A1 (en) * 1994-12-01 1996-06-05 Tosoh Corporation Method of detecting specific nucleic acid sequences
WO1997045539A1 (en) * 1996-05-31 1997-12-04 Mikael Kubista Probe for analysis of nucleic acids
WO1999013105A1 (en) * 1997-09-05 1999-03-18 Mikael Kubista Method for the preparation of a probe for nucleic acid hybridization
WO1999037717A1 (en) * 1998-01-23 1999-07-29 The Perkin-Elmer Corporation Asymmetric cyanine dye quenchers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ILIANA I. ET AL.: "Fluorescence spectral characteristics of novel asymmetric monomethine cyanine dyes in nucleic acid solutions", FEBS LETTERS, vol. 405, 1997, pages 141 - 144 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073118A2 (en) * 2000-03-29 2001-10-04 Lgc (Teddington) Limited Hybridisation beacon and method of rapid sequence detection and discrimination
WO2001073118A3 (en) * 2000-03-29 2002-09-12 Lgc Teddington Ltd Hybridisation beacon and method of rapid sequence detection and discrimination
EP1715063A3 (en) * 2000-03-29 2007-05-02 LGC Limited Hybridisation beacon and method of rapid sequence detection and discrimination
WO2001094473A1 (en) * 2000-06-07 2001-12-13 Lightup Technologies Ab Solid phase synthesis of cyanine dyes
WO2002014555A2 (en) * 2000-08-11 2002-02-21 University Of Utah Research Foundation Single-labeled oligonucleotide probes
WO2002014555A3 (en) * 2000-08-11 2003-02-27 Univ Utah Res Found Single-labeled oligonucleotide probes
US6635427B2 (en) 2000-08-11 2003-10-21 University Of Utah Research Foundation Single-labeled oligonucleotide probes for homogeneous nucleic acid sequence analysis
EP2436780A3 (en) * 2007-10-02 2012-12-19 Charles Brush Monomethine dyes
US8378115B2 (en) 2007-10-02 2013-02-19 Thermo Fisher Scientific (Milwaukee) LLC Monomethine dyes

Also Published As

Publication number Publication date
WO2001002558A8 (en) 2001-04-19
AU6042700A (en) 2001-01-22
SE9902565D0 (en) 1999-07-05

Similar Documents

Publication Publication Date Title
US6329144B1 (en) Probe for analysis of target nucleic acids
US6117973A (en) PNA monomers with electron donor or acceptor
JP4471711B2 (en) Asymmetric cyanine dye quencher
US20030143591A1 (en) Nucleic acid probes and methods to detect and/or quantify nucleic acid analytes
EP0684239A1 (en) A method for detecting a target substance in a sample, utilizing pyrylium compound
CN106459001B (en) Polymethine compound and its purposes as fluorescent marker
KR20170101360A (en) Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same
EP2768306B1 (en) Fluorescent dyes
ES2306265T3 (en) PROBE FOR THE DETECTION OF NUCLEIC ACIDS.
CN101631796B (en) Compound having structure derived from mononucleoside or mononucleotide, nucleic acid, labeling substance, and method and kit for detection of nucleic acid
WO2001002558A1 (en) Probe for analysis of nucleic acids
US20030088109A1 (en) Fluorescent dye
US7786298B2 (en) Compounds and methods for nucleic acid mismatch detection
AU768137B2 (en) Probe for analysis of nucleic acids
EP1362894B1 (en) Method for optical measurement of multi-stranded nucleic acid using cyanine dyes
CA2451442A1 (en) Fluorescent dyes for probes for analysis of nucleic acids
CN111801340A (en) Novel quencher and reporter dye combinations
EP1287077B1 (en) Solid phase synthesis of cyanine dyes
WO2013189043A1 (en) Green fluorescent cyanine dyes, preparation method and uses thereof
US20230332210A1 (en) Reporter and use thereof
US8367323B2 (en) Analytical method involving detection of an exciplex
Deligeorgiev et al. Cyanine dyes as fluorescent non-covalent labels for nucleic acid research
US20130040859A1 (en) Spectrally-resolved chemiluminescent probes for sensitive multiplex molecular quantification
PL190263B1 (en) Probes for use in analysing nucleic acids
Rastede The Design, Synthesis and Characterization of Bright Fluorescent Probes for Biomolecule Detection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C1

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WR Later publication of a revised version of an international search report
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP