Luminescence hybridisation assay method
20230212652 · 2023-07-06
Inventors
Cpc classification
C12Q1/6818
CHEMISTRY; METALLURGY
C12Q1/6818
CHEMISTRY; METALLURGY
International classification
Abstract
This invention relates to a bioassay method for detecting and/or quantitating a short single-stranded nucleic acid analyte employing a binary probe system, where at least one of the two discrete oligonucleotide probe parts of the binary probe has partially double-stranded (self-complementary) stem-loop structure at one terminus and single-stranded overhang sequence region at the other terminus, where the single-stranded terminal regions of both discrete parts of the binary probe hybridize to adjacent complementary regions in the sequence of the nucleic acid analyte molecule, and at least one discrete part of the binary probe comprising a stem-loop structure and single-stranded overhang sequence region hybridizes to terminal region in the sequence of the nucleic acid analyte molecule forming a nick structure. The binary probe system employed in the bioassay method is based on a luminescent reporter technology, either lanthanide chelate complementation or resonance energy transfer with lanthanide label as a donor. Thereby the method allows detection and/or quantitation of the short nucleic acid analyte molecule by time-resolved fluorometry.
Claims
1. A bioassay method for detecting and/or quantitating a nucleic acid (I) contacting two oligonucleotide probes with a sample, wherein (a) a first oligonucleotide probe comprises a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a first region of the nucleic acid analyte, and b) a second oligonucleotide probe comprises a single-stranded terminal sequence that is complementary to and is capable of selectively hybridizing with a second region of the nucleic acid analyte, wherein said first region of the nucleic acid analyte is a first terminal region and said single-stranded terminal sequence overhang of said first oligonucleotide probe hybridizes to said first region of the nucleic acid analyte, hybridization results in formation of a first nick structure between a terminus of said nucleic acid analyte and a first terminus of said first oligonucleotide probe, and said first terminus of said first oligonucleotide probe is part of said double-stranded terminal stem-loop structure of said first oligonucleotide probe, and wherein said first and second region of the nucleic acid analyte are strictly adjacent regions of said nucleic acid analyte, and said single-stranded terminal sequences of said first and second oligonucleotide probe hybridize to said nucleic acid analyte forming a second nick structure between a second terminus of said first oligonucleotide probe and a first terminus of said second oligonucleotide probe; and (II) detecting the presence or absence of said nucleic acid analyte bound to said first and second oligonucleotide probes, wherein the presence of said nucleic acid analyte bound to said first and second oligonucleotide probes confirms the presence of the analyte in the sample.
2. The bioassay method according to claim 1 , wherein said second oligonucleotide probe comprises a double-stranded terminal stem-loop structure and said second region of the nucleic acid analyte is a second terminal region and said single-stranded terminal sequence of second oligonucleotide probe hybridizes to said second region of the nucleic acid analyte, and said hybridization results in formation of a third nick structure between a terminus of said nucleic acid analyte and a second terminus of said second oligonucleotide probe comprising said double-stranded terminal stem-loop structure, and said second terminus of said second oligonucleotide probe is part of said double-stranded terminal stem-loop structure of said second oligonucleotide probe.
3. The bioassay method according to claim 1, wherein either: (a) said first terminuses are 5′ ends (five prime ends) and said second terminuses are 3′ ends (three prime ends), or (b) said first terminuses are 3′ ends (three prime ends) and said second terminuses are 5′ ends (five prime ends).
4. The bioassay method according to claim 1, wherein said nucleic acid analyte is a single stranded nucleic acid with length of 10 - 50 nucleotides.
5. The bioassay method according to claim 1, wherein said nucleic acid analyte is microRNA (miRNA) with length of 17 - 25 nucleotides.
6. The bioassay method according to claim 1, wherein said first and second oligonucleotide probes comprise DNA (deoxyribonucleic acids) or RNA (ribonucleic acids) or any combination of them.
7. The bioassay method according to claim 1, wherein said first and second oligonucleotide probes are both labelled, or said first and second oligonucleotide probes are both labelled and, in addition, either first or second oligonucleotide probe is coupled to any kind of solid support.
8. The bioassay method according to claim 1, wherein said first and second oligonucleotide probes are labelled with a fluorescence resonance energy transfer pair, wherein, either: (a) said first oligonucleotide probe comprises a fluorescent donor and said second oligonucleotide probe comprises a fluorescent acceptor or quencher, or (b) said first oligonucleotide probe comprising a fluorescent acceptor or quencher and said second oligonucleotide probe comprising a fluorescent donor; and wherein resonance energy transfer between said fluorescence resonance energy transfer pair is enabled upon occurrence of both said hybridization events, hybridization of said single-stranded terminal sequence overhang of said first oligonucleotide probe to said first region of the nucleic acid analyte, and hybridization of said single-stranded terminal sequence of said second oligonucleotide probe to said second region of the nucleic acid analyte.
9.The bioassay method according to claim 8,wherein said fluorescence resonance energy donor is a luminescent lanthanide chelate comprising a lanthanide ion.
10.The bioassay method according to claim 1,wherein said first and second oligonucleotide probes are labelled with a switchable lanthanide luminescence label system, wherein, either: (a) said first oligonucleotide probe comprises a lanthanide ion carrier ligand and a lanthanide ion and said second oligonucleotide probe comprises an antenna ligand, or (b) said first oligonucleotide probe comprises an antenna ligand and said second oligonucleotide probe comprises a lanthanide ion carrier ligand and a lanthanide ion; and wherein luminescence of said switchable lanthanide luminescence label system is switched on upon occurrence of both said hybridization events, hybridization of said single-stranded terminal sequence of said first oligonucleotide probe to said first region of the nucleic acid analyte, and hybridization of said single-stranded terminal sequence of said second oligonucleotide probe to said second region of the nucleic acid analyte.
11.The bioassay method according to claim 9, wherein said lanthanide ion is selected from the group consisting of praseodymium(III), neodymium(III), samarium(III), europium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), thulium(III) and ytterbium(III).
12.The bioassay method according to claim 2,wherein a ligase enzyme is used to form a covalent bond in a place of any one or any combination of said first, second and third nick structures formed upon occurrence of any of said hybridization events between said first oligonucleotide probe, said second oligonucleotide probe and the nucleic acid analyte.
13. A kit for detecting and/or quantitating a short nucleic acid analyte molecule, the kit comprising: (a) a first oligonucleotide probe comprising a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a first region of a single-stranded nucleic acid analyte, and b) a second oligonucleotide probe comprising a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a second region of said single-stranded nucleic acid analyte, wherein said first and second region of the single-stranded nucleic acid analyte are strictly adjacent regions of said single-stranded nucleic acid analyte, wherein a terminal nucleotide in a terminus of the single-stranded terminal sequence overhang of said first oligonucleotide probe and a terminal nucleotide in a terminus of the single-stranded terminal sequence overhang of said second oligonucleotide probe are designed to bind to adjacent nucleotides in said single-stranded nucleic acid analyte in order to form a first nick structure between said terminal nucleotides of the single-stranded terminal sequence overhangs of said first and second oligonucleotide probes when said probes are bound to said single-stranded nucleic acid analyte, said first nick structure lacking a phosphodiester bond between a pair of adjacent nucleotides bound to a complementary strand; and wherein said single-stranded terminal sequence overhangs of said first and second oligonucleotide probes are further designed so that when said probes are bound to said single-stranded nucleic acid analyte, one of the terminal nucleotides of said single-stranded nucleic acid analyte forms a second nick structure with a terminal nucleotide in a terminus which is part of said double-stranded terminal stem-loop structure of the first oligonucleotide probe and another of the terminal nucleotides of said single-stranded nucleic acid analyte forms a third nick structure with a terminal nucleotide in a terminus which is part of said double-stranded terminal stem-loop structure of the second oligonucleotide probe, said second and third nick structures lacking a phosphodiester bond between a pair of adjacent nucleotides bound to a complementary strand.
14. The kit according to claim 13, wherein: i) said first oligonucleotide probe comprises a 5′ end at the terminus of the single-stranded terminal sequence overhang and said second oligonucleotide probe comprises a 3′ end at the terminus of the single-stranded terminal sequence overhang; or alternatively ii) said first oligonucleotide probe comprises a 3′ end at the terminus of the single-stranded terminal sequence overhang and said second oligonucleotide probe comprises a 5′ end at the terminus of the single-stranded terminal sequence overhang.
15. The kit according to claim 13, comprising said first and second oligonucleotide probes bound to said single-stranded nucleic acid analyte as a control sample.
16. The kit according to claim 13, wherein said single stranded nucleic acid analyte is with length of 10 - 50 nucleotides.
17. The kit according to claim 13, wherein said single stranded nucleic acid analyte is a microRNA (miRNA), with length of 17 - 25 nucleotides.
18. The kit according to claim 13, wherein said first and second oligonucleotide probes are both labelled, or said first and second oligonucleotide probes are both labelled and, in addition, either first or second oligonucleotide probe is coupled to any kind of solid support.
19. The kit according to claim 18 , wherein said first and second oligonucleotide probes are labelled with a fluorescence resonance energy transfer pair, wherein, either (a) said first oligonucleotide probe comprises a fluorescent donor and said second oligonucleotide probe comprises a fluorescent acceptor or quencher, or (b) said first oligonucleotide probe comprising a fluorescent acceptor or quencher and said second oligonucleotide probe comprising a fluorescent donor; and wherein resonance energy transfer between said fluorescence resonance energy transfer pair is enabled upon occurrence of both said hybridization events, hybridization of said single-stranded terminal sequence overhang of said first oligonucleotide probe to said first region of the nucleic acid analyte, and hybridization of said single-stranded terminal sequence overhang of said second oligonucleotide probe to said second region of the nucleic acid analyte.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0062] Current bioassay methods to detect and/or quantify short nucleic acid targets with defined base sequence and length such as mature microRNAs are limited in specificity and sensitivity. The inventor has found that a nick forming binary probe system based on at least one stem-loop hybridization probe with complementary overhang to the nucleic acid target combined with time-resolved fluorescence resonance energy transfer label pair or formation of lanthanide chelate complex provides a unique approach to sensitive and specific measurement of short sequence nucleic acid analytes such as mature microRNAs with defined nucleobase sequence and length about 22 nt without use of enzymatic nucleic acid amplification or extension.
[0063] In addition to the sensitive measurement of short sequence nucleic acid analytes that is challenging with conventional fluorescence based methods, the invention provides improved specificity to the nucleic acid analytes of defined length, nucleobase sequence composition and also terminal nucleobase sequences. The invention solves how to directly detect and quantify defined short mature microRNAs without interference of other closely related nucleic acids such as pre-miRNA, pri-miRNA and genomic DNA, that can even comprise the same base sequence as part of their longer sequence. The invention further provides high hybridization selectivity to sequence of the target nucleic originating from the base stacking effect, which increases the melting temperature due to formation of one or multiple nick structures in perfectly matching probe hybridization. The formation of the mixed chelate complex is further introduced to increase the stabilization effect based on the co-operative binding of binary probe system.
[0064] Binary stem-loop probe system provides surprising advantages in the detection of short nucleic acid target. First, due to base-stacking effect and nick formation between the target and the probe, it improves the probe affinity and enables sensitive detection of short nucleic acid target that is not possible with linear probes. Second, it provides strict selectivity to the length of product and terminal nucleobase sequence that is not possible with linear probes. Third, the method enables unbiased, direct and amplification free detection of the target in homogeneous assay format, and fourth, the method can also be used for multiplexed array based spatially resolved detection and additional mode of multiplexing is based on the fluorescence color. The stem-loop binary probe concept has not been described even both stem-loop probes (also called hairpin probes) [Nucleic Acids Res 2001; 29: 996-1004] and binary probes as such have been known for decades [Trends in Biotech 2002; 20: 249-256]. In most nucleic acid assays the targets are significantly longer than in the case of mature miRNA and therefore in other applications there is no need to such strict discrimination based on both the target length and sequence composition that is provide by binary stem-loop probe system. The combination of two stem-loop oligonucleotide probes in a stem-loop binary probe makes the system also superior to conventional single-stranded linear probe in target hybridization. The contiguous base stacking interaction [Nucleic Acids Res 2006; 34: 564-574] around the formed nick structures, i.e. between double-stranded stem of the probe and duplex formed with perfectly matched single-stranded target, provide additional free energy minimization, increasing the stability of the resulting probe-target complex. This extra stabilization is lacking in case of mismatched target and, thus, hybridization specificity is improved, i.e. the method provides better discrimination between matched and mismatched target. Further, the hybridization kinetics of the stem loop probes is also faster than with linear probes [Nucleic Acids Res 2001; 29: 996-1004] providing yet another beneficial feature to the present invention. It is surprising, that the stacking interactions of nucleobases have such significant large role in the structural stability of nucleic acids in aqueous solution [Nucleic Acids Res. 1993; 21: 2051-2056] compared to the hydrogen bonds between the bases.
[0065] Conventional fluorescence based assays are not able to provide adequate sensitivity to direct detection of microRNAs and thus the state of the art assays are based on enzymatic nucleic acid amplification steps either utilizing target as template or primer [Lab. Invest. 2019; 99: 452-469]. Time resolved fluorescence based FRET-assays have been demonstrated, but these methods are also amplification based [ACS Nano 2015; 9: 8449-8457] or utilize conventional linear probes and there is no concurrent binding of two oligonucleotide probes to the target as in case of binary probes [Chem. Sci. 2018; 9: 8046-8055].
[0066] In scientific literature a binary stem-loop probe based ligation assay has been earlier described for detection of mature miRNA targets [Talanta 2011; 85: 17560-1765], but it has a major difference to the present invention. The assay is based on formation a nick structure only between the overhangs of the stem-loop probes hybridized on the complementary RNA template and ligation of the nick preferably with T4 RNA ligase 2. The assay utilizes only stem-loop probes that don’t produce nicks between oligonucleotide probe and the target and, thus, don’t benefit of the similar base stacking effect as in the present invention. The nick formation in the assay is actually avoided intentionally by design by adding noncomplementary bases to the end of the stem sequence most likely to avoid the formation of the nick and avoid possible ligation. This is completely in contrast to the present invention and teaches not to utilize the advantage of stem-loop probes in the binary probe hybridization assay. Further, the ligated binary probe is not directly detected based on fluorescence, but the probes act as template for further quantitative PCR reaction that is monitored with double-stranded DNA intercalating dye.
[0067] Molecular beacon type stem-loop oligonucleotide probes [Angew. Chem. Int. Ed. Engl. 2009; 48: 856-870] are widely used luminescence hybridization bioassays, but these are not binary probes and in contrary to the present invention they contain the complementary sequence towards the target nucleic acid in the loop region between the complementary stem regions of the sequence. In the present invention the complementary sequence towards the target nucleic acid fragment is a terminal overhang sequence in the stem-loop oligonucleotide probe and there is no single stranded gap sequence between the one of the complementary stem regions of the probe oligonucleotide sequence and the complementary sequence towards the target, which enables formation of the nick structure upon. Further, in the present invention the stem structures of the probes are not opened upon hybridization.
[0068] The stem-loop based binary probes are able to bind shorter single stranded nucleic acid targets than possible with linear binary probe system. Shorter complementary sequences resulting in higher melting temperature can be used due to stabilization effect of base stacking within double stranded region, when the terminal sequence of the target hybridizes next to double-stranded stem region of the probe (resulting nick structure). The especially suitable targets are short single stranded nucleid acid analytes such microRNAs (miRNA, single stranded, length between 18-24 nt, most commonly 22 nt). When the binary probe comprises a lanthanide chelate complementation label pair, the adjacent binding of the two parts of the stem-loop based binary probe system to the target nucleic acid fragment results in self-assembly of mixed lanthanide chelate complex by coordination binding of the antenna chromophore to the lanthanide ion in the carrier chelate, and renders the binary probe system luminescent. The adjacent hybridization events are either in immediate vicinity, strictly adjacent i.e. 0 nt between the adjacent recognized sequences forming a nick structure, or there is 1 - 20 nt non-recognized sequence gap on target between the proximal recognized sequences. Simultaneous binding of the both parts of the binary probe system to immediately adjacent positions (nick structure formed between the probe overhangs hybridized to the target) and the self-assembly of the mixed lanthanide chelate complex (coordination bonds) strengthen together the melting temperature and improve the specificity of the target detection due to both base stacking effect and antenna ligand coordination to ion carrier chelate.
[0069] Switchable lanthanide luminescence is a sensitive luminescence reporter technology, which has been found superior method for homogeneous nucleic acid hybridization assays. In the present invention it is further combined with the stem-loop binary oligonucleotide probe system to improve the detectability of short single stranded nucleic acid targets such as mature microRNAs. The detection with binary probe system is more specific than with any single probe based systems since simultaneous binding of two discrete parts of the probe system are required for signal generation and the specificity is further enhanced by the formation of nick structures and enhanced affinity provided by base stacking.
[0070] Embodiments of the invention enable thus specific and sensitive detection of mature microRNAs without amplification even from clinical samples with high autofluorescence background due to time-gated fluorescence detection of switchable lanthanide luminescence.
[0071] The alternative embodiments of the invention are described in
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[0076] If the sequence composition of the short target nucleic acid is not fully complementary at the binding regions of the two discrete oligonucleotide probes of the binary probe system or the length of the short target nucleic acid does not allow formation of any of the nick structures, at least one the two discrete oligonucleotide probes of the binary oligonucleotide probe system is not hybridized. When the two discrete oligonucleotide probes are not both hybridized simultaneous on the target nucleic acid, no signal is generated by the binary probe system.
Definitions
[0077] The terms “stem-loop” and “stem-loop structure” shall be understood here to mean a lollipop-shaped nucleic acid structure, that is also known as hairpin structure, formed when a single-stranded nucleic acid molecule comprising an intramolecular palindromic sequence, i.e. the nucleic acid molecule containing the matching sequences in 5′ to 3′ and 3′ to 5′ directions (reverse complement), loops partially back on itself to form a complementary double-stranded region (stem) topped by a single-stranded loop region comprising the nucleotides located between the matching sequences in the nucleic acid molecule.
[0078] The term “overhang” associated with the stem-loop structure shall be understood here to mean single-stranded 5′ or 3′ terminal stretch of nucleic sequence outside the stem-loop structure.
[0079] The term “stem-loop probe” shall be understood here to mean oligonucleotide probe that contains one stem-loop structure and one either 5′ or 3′ terminal overhang sequence that is entirely complementary to the fragment of target nucleic acid. The terminus that is not part of the overhang sequence is part of the double-stranded stem structure. The stem-loop probe is also known as hairpin probe.
[0080] The terms “nick” and “nick structure” shall be understood here to mean a discontinuity in double-stranded nucleic acid, where there is a gap in place of the phosphodiester bond between strictly adjacent nucleotides in one of the two nucleic acid strands of the double helix, but the other strand is intact, and the hybridized strands don’t dissociate even in presence of the nick. In the nick structure there is thus a break in one strand of double-stranded nucleic acid caused by a missing phosphodiester bond between two neighbouring (i.e. immediately adjacent in the nucleic acid sequence) nucleotides, that are both complementary in base pairing to the opposite strand. The nick structure comprises 5′ and 3′ terminus in one strand of double-stranded nucleic acid DNA and, in the opposite strand between the nucleotides complementary in base pairing to the terminal nucleotides of the mentioned 5′ and 3′ terminuses, there is no single stranded region, i.e. not even a single nucleotide without complementary nucleotide in base pairing with the other strand. The nick structure can comprise or not comprise the 5′ terminal phosphate (i.e. phosphate group coupled at 5′ terminal hydroxyl group) at the location of the missing phosphodiester bond. The 3′ terminus at the nick structure comprises typically a 3′ hydroxyl group.
[0081] The term “oligonucleotide probe” refers to labeled polynucleotide that comprises a complementary sequence to the nucleic acid analyte of interest and is used to detect and quantitate the nucleic acid analyte as part of the probe system in the oligonucleotide hybridization assay.
[0082] The term “complementary” and “complementary sequence” shall be understood here to mean nucleic acid sequence of bases that can hybridize and form double-stranded structure with other nucleic acid sequence by matching base pairs (A-T, A-U and C-G). In the double-stranded structure the sequences go in opposite directions, i.e. complementary sequence is reverse complement of the other sequence. Upon hybridization the complementary nucleic acid sequences form a double-stranded nucleic acid structure, typically a DNA double helix or an RNA-DNA duplex. Two single-stranded nucleotide sequences are typically said to be complementary, when the nucleotides of one strand, optimally aligned and compared, pair selectively with at least about 80% of the nucleotides of the other nucleic acid strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. As known to those skilled in the art, a very high degree of complementarity, preferably 100%, is needed for specificity and sensitivity involving hybridization.
[0083] The terms “binary probe”, “binary probe system” and “binary oligonucleotide probe” shall be understood here to mean pair of oligonucleotide probe molecules, i.e. two discrete oligonucleotide probe parts, that produce a detectable signal only when the both parts are hybridized to proximal or adjacent positions in the target nucleic acid molecule. The both parts of the binary probe comprise a oligonucleotide sequence that is complementary to the adjoining positions of the target nucleic acid and, in addition, functional moiety, part of label pair or label fragment that produce a signal when two parts of the binary probe hybridize on the target nucleic acid. Examples of such binary probes are split and two-component probes [Chem. Rev. (2010) 110: 4709-4723].
[0084] The terms “specifically hybridize” and “specific hybridization” and “selectively hybridizing” are used herein to mean the binding, duplexing, or hybridizing of a nucleic acid sequence preferentially to a particular complementary nucleotide sequence under stringent conditions.
[0085] The terms “labeling” and “oligonucleotide labeling” shall be understood here to mean attachment of a label moiety covalently to the oligonucleotide probe at one or multiple defined positions. The labeling of the oligonucleotide probe can be done during oligonucleotide synthesis by using an appropriate building block to introduce the label moiety only or modified nucleotide containing the label moiety to the specific position of the oligonucleotide. [Chapter “Solid-phase oligonucleotide labeling with DOTA” by Jaakkola, et al. (2007) in Current protocols in nucleic acid chemistry, edited by Beaucage, S.L. et al.; Chapter 14: Unit 4.31; online publication by John Wiley & Sons]. Alternatively labeling can be done post synthetically by coupling e.g. amino reactive label moiety to the modified nucleotide, comprising a primary amino group coupled to the nucleotide with a linker, in the synthesized oligonucleotide. In both approaches the label is coupled to oligonucleotide via linker. Combination of both during synthesis and post synthetic labeling enables introduction of different label moieties to distinct position in the oligonucleotide. Modified nucleotide building blocks, where linker is coupled to the nucleobase in a position that base can still properly hybridize to the complementary base, enable attachment of the label practically to any position of the oligonucleotide and double helix without interfering the base stacking and stability of double helix. The linker can be e.g. aliphatic carbon chain or polyethyelene oxide chain. The label moiety can be covalently attached to the using e.g. iodoacetamide, N-hydroxysulfosuccinimide, maleimide or isothiocyanate activation “click-chemistry” approaches [J. Am. Chem. Soc. (2005) 127:14150-14151; Trends Biochem. Sci. (2005) 30:26-34].
[0086] The term “solid-support” refers to an insoluble material where one of the probes can be adsorbed or immobilized utilizing similar coupling chemistries as used for labeling of the oligonucleotide probe. Known materials of this type include hydrocarbon polymers such as polystyrene and polypropylene. In addition, the solid support be composed of silica gel, silicone wafers, glass, metals. The solid support may be physically in the form of particulates, beads, tubes, slides, strips, disks or microtitration wells and plates.
[0087] The terms “fluorescence” and “luminescence” shall be understood here to cover photoluminescence, i.e. luminescence excited by light, fluorescence, including delayed fluorescence with microsecond or millisecond fluorescence lifetime, and phosphorescence. In addition, the term shall cover electrogenerated luminescence and electrochemiluminescence. The luminescence can be measured or imaged either as steady-state luminescence or as time-gated luminescence.
[0088] The terms “lanthanide” and “lanthanide ion” and “luminescent lanthanide ion” and “Ln.sup.3+” shall be understood here to be equivalent to “rare earth metal ion” and to include single trivalent lanthanide ions and any combination of different trivalent lanthanide ion or rare earth ion from the following: cerium, neodymium, praseodymium, samarium, europium, promethium, gadolinium, lutetium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and yttrium, especially europium, terbium, samarium, dysprosium, erbium, neodymium and ytterbium.
[0089] In this disclosure the terms “luminescent lanthanide chelate”, “fluorescent lanthanide chelate”, “luminescent lanthanide complex”, and “complemented lanthanide chelate” shall be understood to include luminescent complexes comprising a lanthanide ion carrier chelate and light-harvesting antenna ligand, where the luminescence of the lanthanide ion in the ion carrier chelate is excited through a light-harvesting chromophore or other excitable structure in the light-harvesting antenna. The luminescent lanthanide chelate can comprise the light harvesting antenna covalently joined to the ion carrier ligand. The complemented lanthanide chelate can be a mixed chelate comprising the ion carrier chelate and the light-harvesting antenna ligand bound to the lanthanide ion by coordination bonds. Examples of intrinsically fluorescent lanthanide chelates are europium(III) and terbium(III) chelates and cryptates that can be excited at wavelength range 320 - 365 nm through the chromophore that is part of the ligand and and emit at 620 nm or 545 nm depending on the lanthanide ion, respectively.
[0090] The terms “lanthanide ion carrier chelate”, “ion carrier chelate” and “carrier chelate” shall be understood to include as such essentially non-luminescent lanthanide chelate complexes and their derivatives, which comprise a chelating ligand, i.e. ion carrier ligand, and a luminescent lanthanide ion, but which do not comprise an efficient light-harvesting antenna chromophore that is essential to efficiently excite the luminescence of the lanthanide ion in the carrier chelate. Examples of lanthanide ion carrier chelates are cyclic or non-cyclic aminopolycarboxylic acid chelates of europium(III) or other luminescent lanthanide ions, where coordination number of the lanthanide ion is preferably equal to or more than 6 dentates, optimally 7 or 8, but which do not contain efficient light-harvesting antenna structure to sensitize and excite the luminescence of the lanthanide ion [PCT Int. Appl. WO 2010/109065]. Additional structures of ion carrier chelates for labelling of an oligonucleotide are illustrated e.g. in US Pat. No. 6,949,639.
[0091] The terms “complementing ligand”, “light harvesting antenna” or “antenna ligand” shall be understood to include as such essentially non-luminescent chelating ligands and their derivatives, which comprise a light-harvesting chromophore or other excitable structure and which are capable of complementing a lanthanide ion carrier chelate to form a luminescent lanthanide complex, where the luminescence of the lanthanide ion in the carrier chelate is excited through non-radiative energy transfer from a light-harvesting chromophore or other excitable structure in the antenna ligand upon either its photoexcitation or electroexcitation. Typically the antenna ligand is a monodentate, bidentate, tridentate or tetradentate ligand, most preferably bidentate or tridentate ligand, the organic light harvesting structure contains aromatic rings or heterocycles, and the light-harvesting structures has a triplet state energy level appropriate for the trivalent lanthanide ion present in the ion carrier chelate. Examples of suitable triplet state energies and light-harvesting structures for lanthanide ions are presented in PCT Int. Appl. WO 2010/109065 and J. Luminescence (1997) 75: 149-169.
[0092] The terms “FRET-pair” and “fluorescent resonance energy transfer donor and acceptor pair” shall be understood to refer to combination of a fluorescent dye (donor) and another fluorescent or non-fluorescent dye (acceptor), where i) the dyes either have spectral overlap between the emission of the donor and absorption of the acceptor to enable Förster type resonance energy-transfer from donor to acceptor, when the dyes are in close proximity, or ii) the acceptor dye is so called universal quencher that does not need spectral overlap to enable energy-transfer from donor to acceptor, when the dyes are in close proximity.
[0093] The terms “non-luminescent” and “non-fluorescent” shall be understood as a property of a light absorbing compound not to produce any or a significant amount of a desired type of luminescence, e.g. long lifetime luminescence, when excited and relaxing from the excited state. In contrast to luminescent compounds, the excited-state energy of a non-luminescent compound is predominantly relaxed via non-radiative pathways, typically producing heat instead of light, or rapid emission instead of slowly decaying emission or the excitation efficiency is weak. The molar extinction coefficient or molar absorptivity of a non-luminescent compound is very low, typically below 10 L mol.sup.-1 cm.sup.-1, or the fluorescence quantum yield of a non-luminescent compound is very poor, typically below 5 percent, or the lifetime of long-lifetime luminescence is shorter than 1 microsecond, typically less than 100 nanoseconds. Examples of non-luminescent compounds are lanthanide chelates, which do not contain a light-harvesting antenna structure for efficient excitation of the lanthanide ion, and light-harvesting antenna ligands, which are not coordinated to lanthanide ions and, thus, not able to produce long lifetime luminescence.
[0094] The terms “lanthanide luminescence” and “luminescence” shall be understood to mean luminescence (i.e. light emission) obtained from emissive relaxation of electronic transitions of lanthanide ion. Lanthanide luminescence can be generated by excitation of the lanthanide ion by direct or indirect light absorption or by electrogenerated chemical excitation.
[0095] The term “chelate” is defined as a coordination complex where a single central metal ion is coordinated to at least one ligand with at least one coordination bond. These complexes may be named by different principles, and names like chelates, supramolecular compounds, complexes and complexones are used. Special types of chelates include e.g. polyaminocarboxylic acids, macrocyclic complexes, crown ethers, cryptates, calixarenes and phorphyrins. The term “mixed chelate” shall be understood as a chelate comprising at least two different ligands coordinated with at least one coordination bond each.
[0096] The terms “time-resolved lanthanide fluorescence”, “time-resolved fluorescence”, “long-lifetime lanthanide luminescence” and “long-lifetime fluorescence” shall be understood here as lanthanide luminescence, where a luminescence lifetime of the luminescent compound is equal to or more than 1 microsecond (the lifetime being calculated as the time wherein luminescence emission intensity decays to the relative value of 1/e, i.e. to approximately 37% of the original luminescence emission intensity). Examples of compounds capable of long-lifetime fluorescence include, but are not limited to, intrinsically fluorescent chelate complexes of europium(III), samarium(III), terbium(III) and dysprosium(III) containing appropriate light-harvesting antenna.
[0097] The terms “light”, “excitation light” and “emission light” shall be understood to cover electromagnetic radiation at wavelengths from 200 nm to 1600 nm. These wavelengths are called ultraviolet light below 400 nm, near-ultraviolet light between 300-450 nm, visible light between 400-750 nm, near-infrared light between 700-1000 nm and infrared light above 700 nm.
[0098] The terms “short-lifetime fluorescence” and “short-lifetime fluorescent compound” shall be understood to cover fluorescence and fluorescent compounds with a luminescence lifetime of less than 1 microsecond, preferably less than 100 nanoseconds. The short-lifetime fluorescence is also referred by conventional fluorescence.
[0099] The terms “electrogenerated luminescence” and “electrochemiluminescence” shall be understood here as lanthanide luminescence produced by electrogenerated chemical excitation using an electrode and applying electric current or voltage to the electrode. Depending on the electrode where the electrochemical reaction producing luminescence occurs the electrochemiluminescence is called cathodic or anodic electrochemiluminescence. Electrogenerated luminescence compounds are compounds capable of anodic or cathodic electrogenerated luminescence. An example of such a compound is hot electron excited 2,6-bis[N,N-bis(carboxymethyl)-aminomethyl]-4-benzoyl phenol-chelated Tb(III) producing green emission (J Alloys Comp 1995; 225: 502-506), but other lanthanide complexes capable of electrogenerated luminescence exist. Electrogenerated luminescence of lanthanide complexes can also be measured using temporal resolution to improve limit of detection.
[0100] In this disclosure, the term “bioassay” shall be understood to refer to detection and/or quantitation of analyte based on fluorescence or luminescence and utilizing probe pair where at least one of the probes contains double-stranded stem-loop structure. The analyte is typically detected and/or measured from a sample or an aliquot of sample, which sample is e.g. a biological or clinical sample..
[0101] The term “homogeneous bioassay” shall be understood to cover bioassays requiring no separation steps. Single or multiple steps of each; addition of reagents, incubation and measurement are the only steps required. The term “separation step” shall be understood to be a step where a labelled bioassay reagent bound onto a solid-phase, such as for example a microparticle or a microtitration well, is separated and physically isolated from the unbound labelled bioassay reagent; for example the microtitration well is washed (liquid is taken out and, to improve the separation, additional liquid is added and the well emptied) resulting in separation of the solid-phase bound labelled bioassay reagent from the labelled bioassay reagent not bound onto the solid-phase.
[0102] The terms “analyte” and “nucleic acid analyte” shall be understood herein as a polynucleotide substance of interest, which is to be measured by the bioassay from the sample. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.
[0103] The terms “sample” and “biological sample” shall be understood to cover various liquid or solid biological samples whereof the analyte is detected, such as serum, blood, plasma, saliva, urine, faeces, seminal plasma, sweat, liquor, amniotic fluid, tissue homogenate, ascites, samples from environmental studies (water and soil samples) and industrial processes (process solutions). The sample can be also product of pre-treatment process of biological sample.
Preferred Embodiments of the Invention
[0104] A typical luminescence hybridization assay method for detecting nucleic acid analyte and/or quantifying nucleic acid analyte concentration according to the invention employs a binary probe system, comprising a step of contacting two oligonucleotide probes with a sample, wherein [0105] (a) a first oligonucleotide probe comprises a double-stranded terminal stem-loop structure and a single-stranded terminal overhang sequence that is complementary to and is capable of selectively hybridizing with a first region of the nucleic acid analyte, and [0106] b) a second oligonucleotide probe comprises a single-stranded terminal sequence that is complementary to and is capable of selectively hybridizing with a second region of the nucleic acid analyte, and said first region of the nucleic acid analyte is terminal region and said single-stranded terminal overhang sequence of said first oligonucleotide probe hybridizes to said first region of the nucleic acid analyte, and said hybridization results in formation of a first nick structure between a terminus of said nucleic acid analyte and a first terminus of said first oligonucleotide probe, and said first terminus of said first oligonucleotide probe is part of said double-stranded terminal stem-loop structure of said first oligonucleotide probe, wherein the first and second region of the nucleic acid analyte are adjacent regions of the nucleic acid analyte, and the single-stranded terminal sequences of the first and second oligonucleotide probe hybridize to the nucleic acid analyte forming a second nick structure between a second terminus of the first oligonucleotide probe and first terminus of the second oligonucleotide probe;
[0107] and detecting the presence or absence of said nucleic acid analyte bound to said first and second oligonucleotide probes, wherein the presence of said nucleic acid analyte bound to said first and second oligonucleotide probes confirms the presence of the analyte in the sample.
[0108] According to another embodiment of the invention also the second oligonucleotide probe comprises a double-stranded terminal stem-loop structure and a single-stranded terminal overhang sequence that is complementary to and is capable of selectively hybridizing with a second region of the nucleic acid analyte, and the second region of the nucleic acid analyte is terminal region and the single-stranded terminal overhang sequence of second oligonucleotide probe hybridizes to the second region of the nucleic acid analyte, and the hybridization results in formation of a third nick structure comprising first terminus of the nucleic acid analyte and second terminus of the second oligonucleotide probe comprising the double-stranded terminal stem-loop structure, and the second terminus of the second oligonucleotide probe is part of the double-stranded terminal stem-loop structure of the second oligonucleotide probe.
[0109] According to the invention the first terminuses are 3′ ends (three prime ends) and the second terminuses are 5′ ends (five prime ends), or the first terminuses are 5′ ends (five prime ends) and the second terminuses are 3′ ends (three prime ends).
[0110] In typical embodiments of the invention, the nucleic acid analyte is a polynucleotide biopolymer composed of nucleotide monomers, typically either deoxyribonucleotides or ribonucleotides, covalently bonded in a linear chain. In a linear polynucleotide chain, the sequence of nucleotides are linked by phosphodiester bonds present between the adjacent nucleotides, and the linear sequence of nucleotides comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide and plurality of internal nucleotides between said terminal nucleotides. The linear polynucleotide chain can be present either in a linear conformation or twisted and/or folded into three-dimensional conformations stabilized by intramolecular noncovalent bonds that can be destabilized by increased temperature.
[0111] In typical embodiments of the invention the nucleic acid analyte is a single stranded nucleic acid with length of 10 - 50 nucleotides, more preferably 15 - 30 nucleotides and most preferably 16 - 26 nucleotides.
[0112] In typical embodiments of the invention the nucleic acid analyte is a microRNA with length of 17 - 25 nucleotides, and in most typical embodies microRNA with length of 21 - 22 nucleotides.
[0113] In preferred embodiments of the invention, the length of the single-stranded terminal overhang sequence of the stem-loop probe oligonucleotide is 5 - 25 nucleotides, more preferably 5 - 20 nucleotides, most preferably 5 - 15 nucleotides.
[0114] In preferred embodiments of the invention the length of double stranded stem sequence in the stem-loop structure is 4 - 16 base pairs, i.e. the palindromic sequence, located on both side of the loop, has length of 4 - 16 nucleotides.
[0115] In preferred embodiments of the invention the length of single stranded loop sequence in the double stranded stem-loop structure is 1 - 12 nucleotides, more preferably 3 - 8 nucleotides, most preferably 4 - 7 nucleotides.
[0116] In preferred embodiments of the invention the first and second oligonucleotide probes comprise DNA (deoxyribonucleic acids), RNA (ribonucleic acids) or their synthetic analogues such as LNA (locked nucleic acids) and PNA (peptide nucleic acid) or any combination of them, and in the most preferred embodiments of the invention the first and second oligonucleotide probes comprise only DNA or RNA nucleotides.
[0117] In typical embodiments of the invention the first and second oligonucleotide probes are both labelled, or both probes are labelled and one of the probes is further coupled to any kind of solid support. The coupling of the stem-loop probe to the solid-support is preferably via modified nucleotide in the the loop sequence. Examples of such immobilized stem-loop probes with single stranded overhang are described in Nucleic Acids Res. 2001; 29: e92 explaining the preferred length of the loop sequence as 7 nucleotides and comprising the NH.sub.2-C6-dT nucleotide in the middle of the loop sequence.
[0118] According to one embodiment of the invention, the target nucleic acid analyte is detected and/or quantified by using agarose gel electrophoresis to separate the the complex formed upon hybridization of the first and second oligonucleotide probes with the analyte and visualizing the formed complex using intercalating dye. The complex formed has different mobility on the agarose gel electrophoresis compared to the individual probes or the analyte due to difference in size and conformation. An example of miRNA detection assay based on unlabeled probe hybridization reaction and agarose gel electrophoretic separation of hybridization complex is described in [Sciences Advances 2019; 5: aau9443].
[0119] According to yet another embodiment of the invention, the target nucleic acid analyte is detected and/or quantified by using melting curve analysis of the complex formed upon hybridization of the first and second oligonucleotide probes with the analyte and double stranded nucleic acid (dsDNA, dsRNA and DNA:RNA-hybrid) binding dye such as SYBR Green I. The dye binds and fluoresces upon binding to double stranded nucleic acid complexes and the melting curve analysis can be used to the confirm the presence of the complex formed with the analyte. An example of miRNA and nucleic acid sequence mutation detection based on unlabeled probe hybridization reaction and melting curve analysis with SYBR Green I is described in [The Analyst 2013: 141: 2384-2387, doi:10.1039/c6an00001k; PloS ONE 2011; 6: e26534].
[0120] In preferred embodiments of the invention the target nucleic acid analyte is detected and/or quantified using fluorescence measurement, more preferably using time-gated fluorescence measurement.
[0121] According to one embodiment of the invention the first and second oligonucleotide probes are labeled with a fluorescence resonance energy transfer pair, wherein, either [0122] (a) the first oligonucleotide probe comprises a fluorescent donor and the second oligonucleotide probe comprises a fluorescent acceptor or quencher, or [0123] (b) the first oligonucleotide probe comprising a fluorescent acceptor or quencher and the second oligonucleotide probe comprising a fluorescent donor; and wherein resonance energy transfer between the fluorescence resonance energy transfer pair is enabled upon occurrence of both the hybridization events, hybridization of the single-stranded terminal sequence of the first oligonucleotide probe to the first region of the nucleic acid analyte, and hybridization the single-stranded terminal sequence of the second oligonucleotide probe to the second region of the nucleic acid analyte.
[0124] According to one embodiment of the invention, when sensitized acceptor emission is measured, the donor and the acceptor are attached during labeling to such positions in the oligonucleotide probes that after hybridization to the nucleic acid analyte the positions of the label carrying nucleotides are preferably separated by at least 4 nucleotides, more preferably by at least 8 nucleotides, but no more than 24 nucleotides.
[0125] The fluorescence resonance energy donor is preferably a luminescent lanthanide chelate comprising a lanthanide ion. Examples of suitable lanthanide donors are Lumi4-Tb cryptate, Eu-TBP (trisbipyridine) cryptate, Eu-W1024 chelate, Eu-W1284 and Eu-W8044 chelates, as well as nonadentate and decadentate Eu(III) chelates described Anal Chem (2003) 75:3193-201 and Inorg Chem. (2013) 52:8461-6.
[0126] The fluorescent acceptor can be selected based on the spectral overlap between the donor emission and acceptor excitation spectrum from a large variety of fluorescent dyes, preferably from short-lifetime fluorescent compounds with excitation and emission at visible or near-infared region of the spectrum (such as ATTO, QXL, Alexa, Bodipy and Cyanine dyes). Examples of suitable fluorescent acceptors to be used in combination with fluorescent Eu(III) or Tb(III) chelate as donor are ATTO 647 and 647N, Alexa Fluor® 647 and 647N, Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7, XL 665 (crosslinked allophycocyanin), Chromeo™ 494, ATTO 490LS, QXL 610, QXL 670, QXL 680, LC red, Quasar 670, and Oyster 645.
[0127] The non-fluorescent quencher is preferably selected from universal quenchers not requiring efficient spectral overlap such as Dabcyl or any of BHQ or QSY quenchers. Examples of suitable non-fluorecent acceptors to be used in combination with fluorescent Eu(III) or Tb(III) chelate as donor are are dabcyl (dimethylaminoazobenzenesulfonic acid), Iowa Black RQ, IRDye QC-1, BHQ-0, BHQ-1, BHQ-2, BHQ-3, QSY 21 and DDQ-II.
[0128] According to another embodiment of the invention the first and second oligonucleotide probes are labelled with a switchable lanthanide luminescence label system, wherein, either [0129] (a) the first oligonucleotide probe comprises a lanthanide ion carrier ligand and a lanthanide ion and the second oligonucleotide probe comprises an antenna ligand, or [0130] (a) the first oligonucleotide probe comprises an antenna ligand and the second oligonucleotide probe comprises a lanthanide ion carrier ligand and a lanthanide ion; and wherein luminescence of the switchable lanthanide luminescence label system is switched on upon occurrence of both the hybridization events, hybridization of the single-stranded terminal sequence of the first oligonucleotide probe to the first region of the nucleic acid analyte, and hybridization of the single-stranded terminal sequence of the second oligonucleotide probe to the second region of the nucleic acid analyte.
[0131] According to one embodiment of the invention, when the two oligonucleotide probes hybridize to adjacent regions in the nucleic acid analyte, the lanthanide ion carrier chelate and the antenna ligand are attached during labeling to such positions in the oligonucleotide probes that after hybridization to the nucleic acid analyte the positions of the label carrying nucleotides are separated by at least one nucleotide, preferably by 2 - 4 nucleotides to sterically favor the self-assembly and formation of the mixed chelate complex.
[0132] The antenna ligand preferably binds weakly to said lanthanide ion, i.e. in typical embodiments of the invention the antenna ligand is either monodentate, bidentate, tridentate or tetradentate. Examples of preferred antenna ligands such as 4-((4-isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid and 4-((4-((4,6-dichloro-1,3,5-triazin-2-yl)amino)phenyl)ethynyl)pyridine-2,6-dicarboxylic acid are described in PCT Int. Appl. WO 2010/109065 and Analyst 2017; 142:2411-2418, respectively.
[0133] In typical embodiments of the invention the ion carrier chelate is pentadentate, hexadentate, heptadentate or octadentate, preferably hexadentate, heptadentate or octadentate. e.g. the lanthanide ion carrier ligand is derived from linear or cyclic chelators, such as EDTA and DTPA or NOTA and DOTA, respectively. Examples of preferred ion carrier ligands such as (2,2′,2″-(10-(3-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid and N1-(4-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.sup.2,N.sup.3,N.sup.3-tetraacetate) are described in PCT Int. Appl. WO 2010/109065.
[0134] The lanthanide ion is preferably selected from the group consisting of praseodymium(III), neodymium(III), samarium(III), europium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), thulium(III) and ytterbium(III).
[0135] According to a yet another embodiment of the invention, more than one different binary probe system, each composed of at least partially different the first and/or second oligonucleotide probes, is used to construct a multiparametric assay and to detect simultaneously multiple different short nucleic acid analytes from the same sample. Typically, each of the different binary probe systems comprises a different lanthanide ion and/or one of the probes of each binary probe system is coupled to spatially separated surface area on any kind of solid support.
[0136] In preferred embodiments of the invention the emission of the fluorescent complex formed, the emission of the fluorescent donor or the sensitized emission of the fluorescent acceptor emission is measured at a wavelength between 400 and 1600 nm.
[0137] In typical embodiments of the invention upon presence of the target nucleic acid the fluorescence measured of the fluorescent complex formed or the sensitized emission of the acceptor is increased, while the fluorescence measured of the fluorescent donor is decreased, and in preferred embodiments of the invention the presence of the target nucleic acid can be detected and quantified without any separation step.
[0138] According to one embodiment of the invention, when one of the probes is coupled to any kind of solid support, a separation step can be included and only the solid-phase bound signal is read to detect and quantify the target nucleic acid.
[0139] In preferred embodiments of the invention the fluorescence is measured with time-gating, i.e. only the delayed fluorescence is observed. Typical time-gating with lanthanide luminescence comprises a delay of at least 10 .Math.s between the end of excitation and start of emission measurement.
[0140] According to one embodiment of the invention the 5′ hydroxyl terminuses of the oligonucleotide probes are non-phosphorylated.
[0141] According to another embodiment of the invention, the 5′ hydroxyl terminus of at least one of oligonucleotide probes is phosphorylated and a ligase enzyme is used to form a covalent bond in a place of any one or any combination of the first, second and third nick structures formed upon occurrence of any of the hybridization events between the first oligonucleotide probe, said second oligonucleotide probe and the nucleic acid analyte. For example T4 RNA ligase 2 is able to catalyze ligation of DNA-fragments on RNA template and some DNA ligases can also efficient join of 3′-OH-terminated RNA to 5′-phosphate-terminated DNA on a DNA template [Biochemistry 1997; 36: 9073-9079.]
[0142] The ligase enzyme treatment is preferably done at lower temperature than the fluorescence measurement to destabilize and preferably dissociate the hybridized probes in case the nicks area not ligated.
[0143] According to yet another embodiment of the invention, the luminescence hybridization assay based on a binary probe system, comprising two oligonucleotide probes, wherein at least one of the probes comprises a stem-loop structure on a binary probe system, is used in in situ hybridization.
[0144] According to yet another embodiment of the invention, the oligonucleotide probe hybridization is carried out in temperature between 15 - 80° C., more preferably between 15 - 60° C. and most preferably between 20 - 40° C. The measurement is done at the same or at different temperature than the oligonucleotide probe hybridization.
[0145] According to yet another embodiment of the invention the oligonucleotide probe hybridization is carried out in presence of 150 mM - 1 M concentration of NaCl. The measurement is done at the same or at different concentration of NaCl than the oligonucleotide probe hybridization.
[0146] According to one embodiment of the invention, the nucleic acid analyte is detected or quantified from body fluids such as plasma and serum. In preferred embodiments of the invention no enzymatic nucleic amplification or nucleic acid extension is used, but the pretreatment of the sample can include e.g. immunocapture or other nucleic acid analyte concentrating step.
[0147] Examples of alternative embodiments of the invention, the luminescence hybridization assay based on a binary probe system, comprising two oligonucleotide probes, wherein at least one of the probes comprises a stem-loop structure and detection and quantitation of the target nucleic acid requires simultaneous binding of the two oligonucleotide probes to the same nucleic acid target molecule, are further described in
[0148]
[0149] The hybridization of the two stem-loop oligonucleotide probes on the described adjacent regions on the target nucleic acid analyte results in formation of three stabilizing nick structures that enable strong co-operative binding of the two probes even they possess only very short complementary sequences. The co-operative binding is further enhanced by the coordination of the light-harvesting antenna ligand to the lanthanide ion in the ion carrier chelate. In absence of any of the factors affecting this co-operative stabilization effect the dissociation of the probes is favored. The highly selective hybridization renders the formation of the fluorescent chelate complex strictly specific to the presence of the defined 3′ and 5′ terminal sequences in and the defined length of the nucleic acid analyte. In case any difference in the terminal sequences exists or the length of the sequence of the nucleic acid analyte does not match the defined length, i.e. the nucleic acid analyte has either deletion of nucleotides in the sequence or comprises a terminal overhang, at least one of the probes is stays dissociated and thus the fluorescence chelate complex is not formed. The specificity of the method to detect and quantify short nucleic acid analytes is thus unique, and the cooperative stabilization effect enables detection of shorter nucleic acid analyte than possible with existing hybridization probe assays or binary probe hybridization assays.
[0150]
[0151] The hybridization of the stem-loop oligonucleotide probe and the linear oligonucleotide probe on the adjacent regions on the nucleic acid analyte results in formation of two stabilizing nick structures that enable strong co-operative binding of the two probes even especially the stem-loop probe possess only very short complementary sequence. In absence of this co-operative stabilization effect the dissociation of the probes is favored. The highly selective hybridization renders the formation of the fluorescent chelate complex strictly specific to the presence of the defined 3′ terminal sequence in the nucleic acid analyte.
[0152]
[0153] The hybridization of the two stem-loop oligonucleotide probes on the described adjacent regions on the target nucleic acid analyte results in formation of two stabilizing nick structures that enable strong co-operative binding of the two probes even the probes possess only very short complementary sequences. The co-operative binding is further enhanced by the coordination of the light-harvesting antenna ligand to the lanthanide ion in the ion carrier chelate. In absence of this co-operative stabilization effect the dissociation of the probes is favored.
[0154]
[0155] The hybridization of the two stem-loop oligonucleotide probes on the described adjacent regions on the target nucleic acid analyte results in formation of three stabilizing nick structures and provides highly selective and sensitive detection of the target nucleic acid equally as in
[0156]
[0157] The hybridization of the two stem-loop oligonucleotide probes on the described adjacent regions on the target nucleic acid analyte results in formation of three stabilizing nick structures and provides highly selective and sensitive detection of the target nucleic acid equally as in
[0158] A further embodiment provided by the present invention is a kit for detecting and/or quantitating a short nucleic acid analyte molecule, the kit comprising: [0159] (a) a first oligonucleotide probe comprising a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a first region of a single-stranded nucleic acid analyte, and [0160] b) a second oligonucleotide probe comprising a double-stranded terminal stem-loop structure and a single-stranded terminal sequence overhang that is complementary to and is capable of selectively hybridizing with a second region of said single-stranded nucleic acid analyte, [0161] wherein said first and second region of the single-stranded nucleic acid analyte are strictly adjacent regions of said single-stranded nucleic acid analyte, [0162] wherein a terminal nucleotide in a terminus of the single-stranded terminal sequence overhang of said first oligonucleotide probe and a terminal nucleotide in a terminus of the single-stranded terminal sequence overhang of said second oligonucleotide probe are designed to bind to adjacent nucleotides in said single-stranded nucleic acid analyte in order to form a first nick structure between said terminal nucleotides of the single-stranded terminal sequence overhangs of said first and second oligonucleotide probes when said probes are bound to said single-stranded nucleic acid analyte, said first nick structure lacking a phosphodiester bond between a pair of adjacent nucleotides bound to a complementary strand; and [0163] wherein said single-stranded terminal sequence overhangs of said first and second oligonucleotide probes are further designed so that when said probes are bound to said single-stranded nucleic acid analyte, one of the terminal nucleotides of said single-stranded nucleic acid analyte forms a second nick structure with a terminal nucleotide in a terminus which is part of said double-stranded terminal stem-loop structure of the first oligonucleotide probe and another of the terminal nucleotides of said single-stranded nucleic acid analyte forms a third nick structure with a terminal nucleotide in a terminus which is part of said double-stranded terminal stem-loop structure of the second oligonucleotide probe, said second and third nick structures lacking a phosphodiester bond between a pair of adjacent nucleotides bound to a complementary strand.
[0164] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.
EXAMPLES
Example 1
Luminescent Hybridization Assay Using Binary Stem-Loop Oligonucleotide Probe System and Chelate Complementation
[0165] Synthetic target-1 DNA oligonucleotide (5′-TAAAGTGCTTATAGTGCAGGTAG-3′; SEQ ID NO: 1), target-2 DNA oligonucleotide (5′-CAAAGTGCTCATAGTGCAG GTAG-3′; SEQ ID NO: 2), target-3 DNA oligonucleotide (5′-CTTAAAGTGCTTATA GTGCAGGTAGAG-3′; SEQ ID NO: 3), target-4 DNA oligonucleotide (5′-AAGTGCTTATAGTGCAGGT-3′; SEQ ID NO: 4), amino-modified probe-C1 oligonucleotide (5′-GTGCTGACCGTAGTACCGGTCAGCACCTACCTGCA(NH.sub.2-C6dC)-3′; SEQ ID NO: 5) and amino-modified probe-C2 oligonucleotide (5′-TA(NH.sub.2-C6dT)AAGCACTTTAGCGTGCAGCCATACTAGGCTGCACGC-3′; SEQ ID NO: 6) were purchased from GeneLink (www.qenelink.com; Hawthorne, NY, USA).
[0166] Probe-C1 oligonucleotide was labelled with non-luminescent Eu.sup.3+ ion carrier chelate ((2,2′,2″-(10-(3-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri(acetate) europium(III); DOTA-Eu (III)) [Analyst (2015) 140: 3960-3968] at the primary amino group residue of modified cytosine with six carbon linker, and probe-C2 oligonucleotide was labelled with light harvesting antenna ligand (4-((isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid; 3d-antenna) [Analyst (2015) 140: 3960-3968] at the primary amino group residue of modified thymine with six carbon linker. Probe-C1 (25 nmol) was incubated with 20-fold molar excess of DOTA-Eu(III) in 50 mM carbonate buffer, pH 9.8, at +37° C. overnight. The reaction volume was 50 .Math.L. For labelling of probe-C2 with 3d-antenna, the 3d-antenna was first dissolved in N,N-dimethylformamide, and probe-C2, 25 nmol, was incubated with 50-fold molar excess of 3d-antenna in 50 mM carbonate buffer, pH 9.8, at +50° C. overnight with slow rotation. The total reaction volume was 110 .Math.L. The purification of labelled probes was carried out with HPLC (instrumentation from SpectraSystem, Thermo Fisher Scientific, Waltham, MA, USA) using a reverse-phase ODS C18 Hypersil column (150 mm long and i.d. of 4.6 mm) from Thermo Fisher Scientific. Purifications were performed using a linear acetonitrile gradient (from 15% to 40% acetonitrile in 30 min) in an aqueous 50 mM triethylammonium acetate buffer pH 7.0 with a flow rate of 0.5 mL min.sup.-1. The collected fractions were evaporated and dried in miVac concentrator (GeneVac, Ipswich, UK) and dissolved again in storage buffer containing 10 mM Tris (pH 7.5), 50 mM NaCl and 10 .Math.M EDTA. Labelled probes were characterized by measuring absorbance readings at 260 nm and 330 nm. Eu.sup.3+ concentration and labeling degree of probe-C1 was measured with DELFIA technology (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland).
[0167] The luminescence hybridization assay was performed by using low Fluorescence 96-well Maxisorp microtitration plates purchased from Nunc (Roskilde, Denmark) in assay buffer containing 50 mM Tris-HCl (pH 7.75), 600 mM NaCl, 0.1% Tween 20, 0.05% NaN.sub.3, and 30 .Math.M diethylenetriaminepentaacetic acid (DTPA). The probe-C1 and probe-C2 were first denatured separately in 0.2 mL tubes by heating for 2 min at 98° C. and then rapidly cooled in 1 min to room temperature using PTC-200 DNA-Engine (MJ Research, Waltham, MA). The probe-C1 (20 nM) and probe-C2 (20 nM) the target-1 oligonucleotide (0-20 nM) were combined in a total volume of 60 .Math.L and added to the microtitration wells. In addition, in separate control experiments target-1 was replaced with target-2 and target-3 oligonucleotides (20 nM). The plate was incubated first at slow shaking for a short period of time and then without shaking for 15 and 60 minutes at room temperature. Time-resolved fluorescence measurements were made with a 1420 Victor Multilabel Counter (Perkin-Elmer Life And Analytical Life Sciences) by using a 340 nm excitation filter, 615 nm emission filter (with 8 nm fwhm, full-width-at-half-maximum), 400 .Math.s delay and 400 .Math.s measurement time, and counting 1000 measurement cycles.
[0168] The experiment resulted in increasing time-resolved luminescence signal at 615 nm with increasing concentration of target-1 with limit of detection (3×SD) below 100 pM concentration, while target-2, target-3 or target-4 at 20 nM concentration produced no significant difference compared to background signal. This illustrated, that the assay is highly selective to target nucleic acid sequence composition as already few mismatches (target-2) disrupt the signal, and also to the exact length of the target nucleic acid is required, as no signal was generated when the target nucleic acid with longer (target-3) or shorter length (target-4) was tested. The hybridization of the probe pair requires thus, that the nick structures are formed upon probe hybridization at the both termini of the target nucleic acid and the base stacking effect is present. The luminescence hybridization assay according to the invention is thus highly selective: it is strongly dependent on the sequence of the target nucleic acid at both terminuses and the length of the target nucleic acid and any variation in length and/or sequence composition will disrupt the hybridization and and signal generation via chelate complementation.
Example 2
Luminescent Hybridization Assay Using Binary Stem-Loop Oligonucleotide Probe System and Fluorescence Resonance Energy Transfer
[0169] Synthetic target-1 DNA oligonucleotide (5′-TAAAGTGCTTATAGTGCAGGTAG-3′; SEQ ID NO: 1), target-2 DNA oligonucleotide (5′-CAAAGTGCTCATAGTGCAG GTAG-3′; SEQ ID NO: 2), target-3 DNA oligonucleotide (5′-CTTAAAGTGCTTATA GTGCAGGTAGAG-3′; SEQ ID NO: 3), target-4 DNA oligonucleotide (5′-AAGTGCTTATAGTGCAGGT-3′; SEQ ID NO: 4), amino-modified probe-F1 oligonucleotide (5′-GTGCTGACCGTAGTACCGGTCAGCACCTA(NH.sub.2-C6dC)CT GCAC-3′; SEQ ID NO: 5) and amino-modified probe-F2 oligonucleotide (5′-TATAAG(NH.sub.2-C6dC)ACTTTAGCGTGCAGCCATACTAGGCTGCACGC-3′; SEQ ID NO: 6) were purchased from GeneLink (www.genelink.com).
[0170] Probe-F1 oligonucleotide was labelled with intrinsically luminescent Eu.sup.3+ chelate, { 2,2′,2″,2‴-{[4-[(4-iso-thiocyanatophenyl)ethynyl]pyridine-2,6-diyl]-bis(methylene nitrilo)}-tetrakis(acetato)}europium(III) (7d-Eu) [J. Phys. Chem. B (2011) 115: 13685-13694] at the primary amino group residue of modified cytosine with six carbon linker, and probe-F2 oligonucleotide was labelled with Alexa Fluor 680 (succinimidyl ester dye; AF680; Thermo Fisher Scientific) at the primary amino group residue of modified cytosine with six carbon linker. Probe-F1 (5 nmol) and 60-fold molar excess of 7d-Eu were dissolved in a total volume of 30 .Math.L of 50 mM carbonate buffer pH 9.8. The labeling reaction was incubated overnight at +37° C. protected from light. Probe-F2 (5 nmol) was dissolved with a 10-fold molar excess of the AF680 into 50 .Math.L of 100 mM carbonate buffer pH 9.2. The labeling reaction was incubated overnight at +37° C. in a 6 rpm rotation protected from light. The labeled probe oligonucleotides were purified with reverse-phase HPLC (SpectraSYSTEM, Thermo Fisher Scientific) with reverse-phase ODS C18 Hypersil column (150 mm long and i.d. of 4.6 mm) from Thermo Fisher Scientific using a linear acetonitrile gradient in an aqueous 50 mM triethylammonium acetate buffer pH 7.0 and flow rate of 0.5 mL min.sup.-1. The collected fractions were evaporated and dried in miVac concentrator (GeneVac) and dissolved again in storage buffer containing 10 mM Tris (pH 8.0), 50 mM NaCl and 10 .Math.M EDTA. Labelled probes were characterized by measuring absorbance readings at 260 nm, 330 nm and 680 nm. Eu.sup.3+ concentration and labeling degree of probe-F1 was measured with DELFIA technology (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland).
[0171] The luminescence hybridization assay was performed by using low Fluorescence 96-well Maxisorp microtitration plates purchased from Nunc in assay buffer containing 50 mM Tris-HCl (pH 7.75), 600 mM NaCl, 0.1% Tween 20, and 0.05% NaN.sub.3. The probe-F1 and probe-F2 were first denatured separately in 0.2 mL tubes by heating for 2 min at 98° C. and then rapidly cooled in 1 min to room temperature using PTC-200 DNA-Engine (MJ Research, Waltham, MA). The probe-C1 (20 nM) and probe-C2 (20 nM) the target-1 oligonucleotide (0-20 nM) were combined in a total volume of 60 .Math.L and added to the microtitration wells. In addition, in separate control experiments target-1 was replaced with target-2 and target-3 oligonucleotides (20 nM). The plate was incubated first at slow shaking for a short period of time and then without shaking for 15 and 60 minutes at room temperature. Time-resolved fluorescence measurements were made with a 1420 Victor Multilabel Counter (Perkin-Elmer Life And Analytical Life Sciences) by using a 340 nm excitation filter, 730 nm emission filter (with 10 nm fwhm), 60 .Math.s delay and 80 .Math.s measurement time, and counting 1000 measurement cycles.
[0172] The experiment resulted in increasing time-resolved luminescence signal at 730 nm with increasing concentration of target-1 with limit of detection (3×SD) below 200 pM concentration, while target-2, target-3 or target-4 at 20 nM concentration produced no significant difference compared to background signal. The luminescence hybridization assay based on binary stem-loop FRET-probe pair shows thus similar advantages than the equivalent assay based on chelate complementation probe pair. This indicates that the luminescence hybridization assay according to the present invention is highly specific to the target sequence and length, and is able to quantitatively detect small concentration of short target nucleic acid molecules that are otherwise hard to measure.
OTHER PREFERRED EMBODIMENTS
[0173] It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.