FIELD OF INVENTION

The present invention relates to a method for qualitatively or quantitatively detection of oligonucleotides of interest using a fluorescently labelled LNA oligonucleotide probe. The invention also relates to the LNA oligonucleotides probes and a kit for use in analysis of a target oligomer.

BACKGROUND

Antisense oligonucleotides, siRNA and aptamers are being developed as therapeutic agents. The qualitative and quantitative detection of these oligonucleotides in samples like cell cultures, tissue, blood, plasma or urine is a prerequisite to assess their use and to monitor their intracellular uptake, bio-distribution, metabolism and stability in vivo and/or in vitro.

The prior art contains a number of methods for detecting short oligonucleotides in samples that are suspected of containing the oligonucleotide, e.g. as the result of in vitro treatment of a cell culture or in vivo treatment of an animal with the oligonucleotide.

WO 96/06189 describes the qualitative and quantitative detection of oligonucleotides by capillary gel electrophoresis.

Quantitative detection of oligonucleotides using HPLC followed by UV detection is described by Raynaud et al, 1997, The Journal of Pharmacology and Experimental Therapeutics Vol. 281 pp. 420-427.

WO 2008/046645 describing the use of LNA probes in a RT-PCR-based oligonucleotide detection assay.

WO 2010/043512 describes the use of a fluorescently labelled PNA oligonucleotide probe for detection and quantification of oligonucleotides.

The current methods have various draw backs either with respect to sensitivity, complexity in terms of steps needed and limitations to probe design.

OBJECTIVE OF THE INVENTION

The present invention provides a reproducible and quick method for quantitative detection of oligonucleotides to very low levels with probes that tolerate purine-rich stretches.

SHORT DESCRIPTION OF THE FIGURES

Figure 1 shows A) the slope of regression and the correlation of all the data points from the detection of AS01 in cynomolgus monkey plasma. B) shows the slope of regression and the correlation of the data points in the area indicated with a black square of A. Figure 2 shows the chromatograms for the quantization of AS01 using fluorescence detection with LNA-P1 (A-C) and UV detection (D-F). The concentration of AS01 was 1667 ng/ml (A and D), 833 ng/ml (B and E) and 417 ng/ml (C and F). The peak to the right in the chromatogram contains AS01 ( the oligonucleotide of interest) to be quantified.

Figure 3 shows A) the slope of regression and the correlation of all the data points from the detection of AS02 cynomolgus monkey plasma. B) shows the slope of regression and the correlation of the data points in the area indicated with a black square of A.

Figure 4 shows the chromatograms for the quantization of AS02 using fluorescence detection with LNA-P2 (A-C at the following AS02 concentration, 250 ng/ml, 50 ng/ml and 10 ng/ml, respectively) and UV detection (D-F at 10.000 ng/ml, 1250 ng/ml and 625 ng/ml, respectively).

DEFINITIONS Oligonucleotide

The term "oligonucleotide" as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.

Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide to be detected by the method of the present invention (oligonucleotide of interest) is man-made, and is chemically synthesized. The oligonucleotide of interest has typically been purified or isolated and then administered to cell culture or to an animal. The oligonucleotide of interest may be an antisense oligonucleotide, siRNA, aptamer or spiegelmers or a mixture of such oligonucleotides. The oligonucleotide of interest may comprise one or more modified nucleosides or nucleotides. Oligonucleotide metabolite

The term "oligonucleotide metabolite" includes oligonucleotides from which 1 or more nucleotides are deleted from the 3' and / or the 5' end, or which have been cleaved by for example a nuclease such as RNaseH. An oligonucleotide metabolite can also be the metabolism of a compound comprising the oligonucleotide of interest, such as the cleavage of an oligonucleotide conjugate.

Antisense oligonucleotides

The term "Antisense oligonucleotide" as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs. Preferably, the antisense oligonucleotides of the present invention are single stranded. siRNA

The term "siRNA" refers to a double stranded RNA molecule that is capable of blocking gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).

Nucleotides

Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in

nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as "units" or "monomers".

Modified nucleoside

The term "modified nucleoside" or "nucleoside modification" as used herein refers to

nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term "nucleoside analogue" or modified "units" or modified "monomers". Modified internucleoside linkage

The term "modified internucleoside linkage" is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. Nucleotides with modified internucleoside linkage are also termed

"modified nucleotides". In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of interest, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides.

In an embodiment, the oligonucleotide of interest comprises one or more internucleoside linkages modified from the natural phosphodiester to a linkage that is for example more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are modified. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.

Modified internucleoside linkages may be selected from the group comprising phosphorothioate, diphosphorothioate and boranophosphate. In preferred embodiments, the modified

internucleoside linkages are phosphorothioate. Preferably, all the internucleoside linkages in the oligonucleotide of interest are phosphorothioate linkages.

Modified oligonucleotide

The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar- modified nucleosides and/or modified internucleoside linkages . The term chimeric"

oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.

Complementarity

The term complementarity describes the capacity for Watson-Crick base-pairing of

nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non- modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1 .4.1 ).

The term "% complementary" as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide of interest) which, at a given position, are complementary to (i.e. form Watson Crick base pairs with) a contiguous nucleotide sequence, at a given position of a separate nucleic acid molecule (e.g. the LNA oligonucleotide probe). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences, dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.

The term "fully complementary", refers to 100% complementarity. Hybridization

The term "hybridizing" or "hybridizes" as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide of interest and LNA oligonucleotide probe) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T _m ) defined as the temperature at which half of the oligonucleotides of interest are duplexed with the LNA oligonucleotide probe.

The conditions needed for hybridization between the oligonucleotide of interest and the LNA oligonucleotide probe will depend on the affinity. The skilled person will know how to determine the optimal hybridization conditions for the LNA oligonucleotide probe with the oligonucleotide of interest.

The term "hybridized moieties" refers to any oligonucleotide of interest or any of its metabolites which are hybridized to the LNA oligonucleotide probe whereas "unhybridized moieties" refer to any oligonucleotide of interest or any of its metabolites which are not hybridized to the LNA oligonucleotide probe.

Target molecule

A target molecule is the molecule on which the effect of the oligonucleotide of interest can be measured. The target molecule can be a target nucleic acid, a protein encoded by the target nucleic acid, or a protein, peptide, carbohydrate, vitamin or lipid to which the oligonucleotide of interest can bind as such.

Target nucleic acid

A target nucleic acid is an intended target for the oligonucleotide of interest, and may for example be a gene, a RNA, a mRNA, and pre-mRNA, a mature mRNA or a cDNA sequence. For in vivo or in vitro application, the oligonucleotide of interest is typically capable of modulating the expression of the intended target nucleic acid in a cell which is expressing the target nucleic acid. The target nucleic acid may, in some embodiments, be a RNA or DNA, such as micro RNA, non-coding RNA, viral RNA/DNA or a messenger RNA, such as a mature mRNA or a pre-mRNA. An oligonucleotide of interest directed towards a target nucleic acid may also be termed a target oligonucleotide. Target Cell

The term a target cell as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.

High affinity modified nucleosides

A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T ^m ). A high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to +12°C, more preferably between +1 .5 to +10°C and most preferably between+3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2' substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

Sugar modifications

The oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of target oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by

replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO201 1/017521 ) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2', 3', 4' or 5' positions.

Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into target oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.

2' modified nucleosides.

A 2' sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle, and includes 2' substituted nucleosides and LNA (2' - 4' biradicle bridged) nucleosides. For example, the 2' modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2' substituted modified nucleosides are 2'-0-alkyl-RNA, 2'-0- methyl-RNA, 2'-alkoxy-RNA, 2'-0-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293- 213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2' substituted modified nucleosides.

2'-0- e 2'F-RNA 2'F-ANA

2'-O-M0E 2'-0-AHyl 2' -θΈΐΡ Μηβ

The LNA oligonucleotide probe of the invention may in addition to the LNA nucleosides also comprise one or more 2'-substituted modified nucleosides. Locked Nucleic Acid Nucleosides (LNA).

LNA nucleosides are modified nucleosides which comprise a linker group (referred to as a biradicle or a bridge) between C2' and C4' of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.

In some embodiments, the modified nucleoside or the LNA nucleosides of the oligonucleotide of interest and/or the LNA oligonucleotide probe has a general structure of the formula I or II:

Formula I Formula II

wherein W is selected from -0-, -S-, -N(R ^a )-, -C(R ^a R ^b )-, such as, in some embodiments -0-; B designates a nucleobase or modified nucleobase moiety;

Z designates an internucleoside linkage to an adjacent nucleoside, or a 5'-terminal group; Z ^* designates an internucleoside linkage to an adjacent nucleoside, or a 3'-terminal group; X designates a group selected from the list consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, - C(R ^a )=N-, -0-, -Si(R ^a ) ₂ -, -S-, -S0 ₂ -, -N(R ^a )-, and >C=Z

In some embodiments, X is selected from the group consisting of: -0-, -S-, NH-, NR ^a R ^b , -CH ₂ -, CR ^a R ^b , -C(=CH ₂ )-, and -C(=CR ^a R ^b )-

In some embodiments, X is -O-

Y designates a group selected from the group consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, - C(R ^a )=N-, -0-, -Si(R ^a ) ₂ -, -S-, -SO ₂ -, -N(R ^a )-, and >C=Z

In some embodiments, Y is selected from the group consisting of: -CH ₂ -, -C(R ^a R ^b )-, - CH ₂ CH ₂ -, -C(R ^a R ^b )-C(R ^a R ^b )-, -CH ₂ CH ₂ CH ₂ -, -C(R ^a R ^b )C(R ^a R ^b )C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, and -C(R ^a )=N-

In some embodiments, Y is selected from the group consisting of: -CH ₂ -, -CHR ^a -, - CHCH3-, CR ^a R ^b - or -X-Y- together designate a bivalent linker group (also referred to as a radicle) together designate a bivalent linker group consisting of 1 , 2, 3 or 4 groups/atoms selected from the group consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, -C(R ^a )=N-, -0-, -Si(R ^a ) ₂ -, -S-, -S0 ₂ -, -N(R ^a )-, and >C=Z,

In some embodiments, -X-Y- designates a biradicle selected from the groups consisting of: -X-CH ₂ -, -X-CR ^a R ^b -, -X-CHR ^a -, -X-C(HCH ₃ ) ^~ , -0-Y-, -0-CH ₂ -, -S-CH ₂ -, -NH-CH ₂ -, -O- CHCH ₃ -, -CH ₂ -0-CH ₂ , -0-CH(CH ₃ CH ₃ )-, -0-CH ₂ -CH ₂ -, OCH ₂ -CH ₂ -CH ₂ -,-0-CH ₂ OCH ₂ -, - 0-NCH ₂ -, -C(=CH ₂ )-CH ₂ -, -NR ^a -CH ₂ -, N-0-CH ₂ , -S-CR ^a R ^b - and -S-CHR ^a -.

In some embodiments -X-Y- designates -0-CH ₂ - or -0-CH(CH ₃ )-.

wherein Z is selected from -0-, -S-, and -N(R ^a )-, and R ^a and, when present R ^b , each is independently selected from hydrogen, optionally substituted C _ ₆ -alkyl, optionally substituted C ₂ - ₆ -alkenyl, optionally substituted C ₂ - ₆ -alkynyl, hydroxy, optionally substituted Ci-e-alkoxy, C ₂ - ₆ -alkoxyalkyl, C ₂ - ₆ -alkenyloxy, carboxy, Ci _-6 - alkoxycarbonyl, Ci- ₆ -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C _1-6 - alkyl)amino, carbamoyl, mono- and di(C ₁ . ₆ -alkyl)-amino-carbonyl, amino-Ci-e-alkyl- aminocarbonyl, mono- and di(C ₁ . ₆ -alkyl)amino-C ₁ . ₆ -alkyl-aminocarbonyl, d-e-alkyl- carbonylamino, carbamido, d-e-alkanoyloxy, sulphono, d-e-alkylsulphonyloxy, nitro, azido, sulphanyl, d-e-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R ^a and R ^b together may designate optionally substituted methylene (=CH ₂ ), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation.

wherein R ¹ , R ² , R ³ , R ⁵ and R ^5* are independently selected from the group consisting of:

hydrogen, optionally substituted C _ ₆ -alkyl, optionally substituted C ₂ - ₆ -alkenyl, optionally substituted C ₂ - ₆ -alkynyl, hydroxy, Ci-e-alkoxy, C ₂ - ₆ -alkoxyalkyl, C ₂ - ₆ -alkenyloxy, carboxy, Ci _-6 - alkoxycarbonyl, Ci- ₆ -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci _-6 - alkyl)amino, carbamoyl, mono- and di(Ci _-6 -alkyl)-amino-carbonyl, amino-Ci- ₆ -alkyl- aminocarbonyl, mono- and di(Ci- ₆ -alkyl)amino-Ci- ₆ -alkyl-aminocarbonyl, Ci _-6 -alkyl- carbonylamino, carbamido, Ci- ₆ -alkanoyloxy, sulphono, Ci- ₆ -alkylsulphonyloxy, nitro, azido, sulphanyl, Ci _-6 -alkylthio, halogen, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene.

In some embodiments R ¹ , R ² , R ³ , R ⁵ and R ^5* are independently selected from Ci_ ₆ alkyl, such as methyl, and hydrogen.

In some embodiments R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen.

In some embodiments R ¹ , R ² , R ³ , are all hydrogen, and either R ⁵ and R ^5* is also hydrogen and the other of R ⁵ and R ^5* is other than hydrogen, such as C _1-6 alkyl such as methyl.

In some embodiments, R ^a is either hydrogen or methyl. In some embodiments, when present, R ^b is either hydrogen or methyl.

In some embodiments, one or both of R ^a and R ^b is hydrogen

In some embodiments, one of R ^a and R ^b is hydrogen and the other is other than hydrogen In some embodiments, one of R ^a and R ^b is methyl and the other is hydrogen

In some embodiments, both of R ^a and R ^b are methyl. In some embodiments, the biradicle -X-Y- is -0-CH ₂ -, W is O, and all of Ft ¹ , Ft ² , Ft ³ , Ft ⁵ and Ft ^5* are all hydrogen. Such LNA nucleosides are disclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160 which are all hereby incorporated by reference, and include what are commonly known as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides. In some embodiments, the biradicle -X-Y- is -S-CH ₂ -, W is O, and all of Ft ¹ , Ft ² , Ft ³ , Ft ⁵ and Ft ^5* are all hydrogen. Such thio LNA nucleosides are disclosed in WO99/014226 and

WO2004/046160 which are hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is -NH-CH ₂ -, W is O, and all of Ft ¹ , Ft ² , Ft ³ , Ft ⁵ and Ft ^5* are all hydrogen. Such amino LNA nucleosides are disclosed in WO99/014226 and

WO2004/046160 which are hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is -0-CH ₂ -CH ₂ - or -0-CH ₂ -CH ₂ - CH ₂ -, W is O, and all of Ft ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such LNA nucleosides are disclosed in

WO00/047599 and Morita et al, Bioorganic & Med.Chem. Lett. 12 73-76, which are hereby incorporated by reference, and include what are commonly known as 2'-0-4'C-ethylene bridged nucleic acids (ENA).

In some embodiments, the biradicle -X-Y- is -0-CH ₂ -, W is O, and all of R ¹ , R ² , R ³ , and one of R ⁵ and R ^5* are hydrogen, and the other of R ⁵ and R ^5* is other than hydrogen such as C1-6 alkyl, such as methyl. Such 5' substituted LNA nucleosides are disclosed in WO2007/134181 which is hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is -0-CR ^a R ^b -, wherein one or both of R ^a and R ^b are other than hydrogen, such as methyl, W is O, and all of R ¹ , R ² , R ³ , and one of R ⁵ and R ^5* are hydrogen, and the other of R ⁵ and R ^5* is other than hydrogen such as C1-6 alkyl, such as methyl. Such bis modified LNA nucleosides are disclosed in WO2010/077578 which is hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- designate the bivalent linker group -O-

CH(CH ₂ OCH ₃ )- (2' O-methoxyethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81 ). In some embodiments, the biradicle -X-Y- designate the bivalent linker group -0-CH(CH ₂ CH ₃ )- (2'O-ethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81 ). In some embodiments, the biradicle -X-Y- is -0-CHR% W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such 6' substituted LNA nucleosides are disclosed in W010036698 and WO07090071 which are both hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is -0-CH(CH ₂ OCH ₃ )-, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such LNA nucleosides are also known as cyclic MOEs in the art (cMOE) and are disclosed in WO07090071 . In some embodiments, the biradicle -X-Y- designate the bivalent linker group -0-CH(CH ₃ )-. - in either the R- or S- configuration. In some embodiments, the biradicle -X-Y- together designate the bivalent linker group -0-CH ₂ -0-CH ₂ - (Seth at al., 2010, J. Org. Chem). In some

embodiments, the biradicle -X-Y- is -0-CH(CH ₃ )-, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such 6' methyl LNA nucleosides are also known as cET nucleosides in the art, and may be either (S)cET or (R)cET stereoisomers, as disclosed in WO07090071 (beta-D) and WO2010/036698 (alpha-L) which are both hereby incorporated by reference).

In some embodiments, the biradicle -X-Y- is -0-CR ^a R ^b -, wherein in neither R ^a or R ^b is hydrogen, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. In some embodiments, R ^a and R ^b are both methyl. Such 6' di-substituted LNA nucleosides are disclosed in WO

2009006478 which is hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is -S-CHR% W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such 6' substituted thio LNA nucleosides are disclosed in W01 1 156202 which is hereby incorporated by reference. In some 6' substituted thio LNA embodiments R ^a is methyl.

In some embodiments, the biradicle -X-Y- is -C(=CH2)-C(R ^a R ^b )-, such as -C(=CH ₂ )-CH ₂ - , or - C(=CH ₂ )-CH(CH ₃ )-W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. Such vinyl carbo LNA nucleosides are disclosed in WO08154401 and WO09067647 which are both hereby incorporated by reference.

In some embodiments the biradicle -X-Y- is -N(-OR ^a )-, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. In some embodiments R ^a is C - ₆ alkyl such as methyl. Such LNA nucleosides are also known as N substituted LNAs and are disclosed in WO2008/150729 which is hereby incorporated by reference. In some embodiments, the biradicle -X-Y- together designate the bivalent linker group -0-NR ^a -CH ₃ - (Seth at al., 2010, J. Org. Chem). In some embodiments the biradicle -X-Y- is -N(R ^a )-, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. In some embodiments R ^a is C _-6 alkyl such as methyl.

In some embodiments, one or both of R ⁵ and R ^5* is hydrogen and, when substituted the other of R ⁵ and R ^5* is C _1-6 alkyl such as methyl. In such an embodiment, R ¹ , R ² , R ³ , may all be hydrogen, and the biradicle -X-Y- may be selected from -0-CH2- or -0-C(HCR ^a )-, such as -0-C(HCH3)-. In some embodiments, the biradicle is -CR ^a R ^b -0-CR ^a R ^b -, such as CH ₂ -0-CH ₂ -, W is O and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. In some embodiments R ^a is 0 _-6 alkyl such as methyl. Such LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO2013036868 which is hereby incorporated by reference.

In some embodiments, the biradicle is -0-CR ^a R ^b -0-CR ^a R ^b -, such as 0-CH ₂ -0-CH ₂ -, W is O and all of R ¹ , R ² , R ³ , R ⁵ and R ^5* are all hydrogen. In some embodiments R ^a is Ci_ ₆ alkyl such as methyl. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is hereby incorporated by reference.

It will be recognized than, unless specified, the LNA nucleosides may be in the beta-D or alpha- L stereoisoform.

Certain examples of LNA nucleosides are presented in Scheme 1 .

Scheme 1

As illustrated in the examples, in some embodiments of the invention the LNA nucleosides in the oligonucleotide of interest and/or in the LNA oligonucleotide probe are beta-D-oxy-LNA nucleosides. Peptide nucleic acid (PNA)

PNA's are nucleosides where the ribose ring has been substituted with a peptide-like backbone that serves to link the nucleobases together with approximately the same distance as in DNA or

PNA oligonucleotide

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 /23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.

Gapmer

The term gapmer as used herein refers to an antisense oligonucleotide of interest which comprises a region of RNase H recruiting oligonucleotides (gap) which is flanked 5' and 3' by one or more affinity enhancing modified nucleosides (flanks or wings). Various gapmer designs are described herein. Generally, RNaseH recruitment requires a gap 4 to 5 contiguous DNA nucleoside or more. Headmers and tailmers are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e. only one of the ends of the oligonucleotide comprises affinity enhancing modified nucleosides. For headmers the 3' flank is missing (i.e. the 5' flank comprises affinity enhancing modified nucleosides) and for tailmers the 5' flank is missing (i.e. the 3' flank comprises affinity enhancing modified nucleosides).

LNA Gapmer

The term LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside.

Mixed Wing Gapmer

The term mixed wing gapmer refers to a LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2' substituted modified nucleoside, such as, for example, 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'- alkoxy-RNA, 2'-0-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA and 2'-F-ANA nucleoside(s). In some embodiments the mixed wing gapmer has one flank which comprises LNA nucleosides (e.g. 5' or 3') and the other flank (3' or 5' respectfully) comprises 2' substituted modified nucleoside(s). Conjugate

The term conjugate as used herein refers to an oligonucleotide of interest which is covalently linked to a non-nucleotide moiety.

Conjugation of the oligonucleotide of interest to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. At the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs. WO 93/07883 and WO 2013/033230 provide suitable conjugate moieties, which are hereby incorporated by reference. Further suitable conjugate moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr). In particular tri-valent N-acetylgalactosamine conjugate moieties are suitable for binding to the the ASGPr, see for example WO 2014/076196, WO 2014/207232 and WO 2014/179620 (hereby incorporated by reference).

In an embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.

The LNA oligonucleotide probes of the present invention can be used to detect the metabolism of such conjugates as well as their cellular uptake and bio-distribution.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety, to a first region, e.g. an oligonucleotide. In some embodiments of the invention the conjugate or oligonucleotide conjugate of interest may optionally, comprise a linker region (second region) which is positioned between the oligonucleotide (first region) and the conjugate moiety (third region).

The second region may be a biocleavable linker comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. In one embodiment the biocleavable linker is

susceptible to S1 nuclease cleavage. In a preferred embodiment the nuclease susceptible linker comprises between 1 and 10 nucleosides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably between 2 and 6 nucleosides and most preferably between 2 and 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, such as at least 3 or 4 or 5 consecutive phosphodiester linkages. Preferably the nucleosides are DNA or RNA.

Phosphodiester containing biocleavable linkers are described in more detail in WO

2014/076195 (hereby incorporated by reference).

Alternatively, the second region may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. In some embodiments the second region is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker is a C6 amino alkyl group.

Therapeutic

The term "therapeutic" in relation to an oligonucleotide or oligonucleotide conjugate of interest refers to such compounds that are suitable for treatment of an existing disease (e.g. a disease or disorder diagnosed in a patient), and/or prevention of a disease or disorder, i.e. prophylaxis in a subject. It will therefore recognized that therapeutic as referred to herein can, in some embodiments, be prophylactic. Preferably, such subject is a mammal, most preferably a human patient. DETAILED DESCRIPTION OF THE INVENTION

Disclosed is a method for qualitative and quantitative detection of an oligonucleotide of interest or an oligonucleotide conjugate comprising the oligonucleotide of interest comprising the steps of a) obtaining a sample containing or suspected of containing said oligonucleotide or conjugate, b) forming a hybridization mixture by contacting the sample with a fluorescently labelled LNA oligonucleotide probe which is fully complementary to at least 7 or more contiguous nucleotides of said oligonucleotide of interest, c) separating hybridized moieties formed between said oligonucleotide of interest and LNA oligonucleotide probe from unhybridized moieties by anion exchange high performance liquid chromatography (AIEX-HPLC), and d) qualitatively and/or quantitatively detecting said hybridized moieties by fluorescence spectroscopy. The advantages of the present invention over other published oligonucleotide quantitation methods are the quick and simple sample preparation (e.g. no clean-up procedures or extraction steps are required avoiding variability in analyte recovery), the lack of amplification steps prior to detection (resulting in a quick and simple method), the affinity and solubility of the of LNA oligonucleotide probes, which all contribute to high sensitivity and reproducibility.

Furthermore, the method has high-throughput capability and the capability to detect metabolites in one measurement.

The sample containing or suspected of containing said oligonucleotide of interest can be obtained from cell cultures exposed to the oligonucleotide of interest (in vitro) or from animals that have been administered with the oligonucleotide of interest (in vivo). The samples following in vivo administration can for example be blood, plasma, urine or tissue isolated from the animal. Non-limiting examples of tissue samples are adipose tissue, bone marrow, brain, cartilage, cancer cells, heart, immune cells, intestine, kidney, liver, lung, muscle, neurons and pancreatic cells.

In some embodiments the sample is treated with a proteinase prior to addition of the LNA oligonucleotide probe. Preferably, the proteinase is a serine protease, such as proteinase K. The proteinase activity may be increased by the addition of 0.5-1 % sodium dodecyl sulfate (SDS) or Guanidinium chloride (3 M), Guanidinium thiocyanate (1 M) or urea (4 M). If SDS is used, the proteinase treatment may be followed by precipitation of the SDS with a saturated KCI solution. The proteinase treatment serves to remove proteins from the sample and prevent potential degradation of the oligonucleotides by enzymes present in the sample. The sample may also be subjected to centrifugation, homogenization and/or lysis prior to or simultaneously with proteinase K treatment, in particular if the sample is a tissue sample.

The detection of the oligonucleotide of interest is performed using anion exchange HPLC (AIEX- HPLC). One of the most common anion-exchange materials is diethylamino ethyl (DEAE) bonded support, and it has been extensively used to modify original soft-gel supports, porous microparticular silica, and polymer-based materials. Alternatively nonporous, small-particle resins are available, and provide very fast and efficient separations. There are a number of commercially available AIEX columns available for oligonucleotide purification from for example Dionex, Shodex and Tosoh. Elution buffers and HPLC gradients are selected according to the column selected. In some embodiments the elution buffer comprises varying concentration of Sodium perchlorate (NaCI04) buffered with Tris-HCL. The skilled person in the art will know how to optimise an AIEX-HPLC system for the method of the present invention.

The hybridization of the oligonucleotide of interest with the fluorescently labelled LNA

oligonucleotide probe contributes to high sensitivity and reproducibility of the present invention. The presence of LNA in the LNA oligonucleotide probe increases the affinity of the probe towards the contiguous nucleotides in the oligonucleotide of interest which are complementary to the probe. This results in very stable duplexes between the LNA oligonucleotide probe and the oligonucleotide of interest which leads to a thermodynamically controlled hybridization even in presence of a competing RNA or DNA strand (e.g. from the target of the oligonucleotide of interest or the sense strand in siRNA duplexes). The affinity of the LNA oligonucleotide probe to the oligonucleotide of interest can be optimized by varying the amount of DNA and LNA nucleosides in the probe (see the section on LNA oligonucleotides probes below for particular designs). This significantly increases the flexibility in the design of an LNA oligonucleotide probe compared to a PNA probe, which cannot be mixed with other nucleosides.

A further difference between the LNA probe of the present invention and the PNA probe as describe in WO2010/043512 is that PNA contain an uncharged backbone and have limited solubility whereas LNA that has a charged backbone are very soluble in all relevant water buffer systems. During the anion exchange (AIEX) chromatography the salt concentration is gradually increased this will reduce the solubility of the PNA probe, whereas due to the charged backbone of the LNA probe, an increase in salt will result in an increase in the stability and the melting temperature of the duplex between the LNA oligonucleotide of interest and the LNA

oligonucleotide probe during the AIEX chromatography. This, in combination with the high binding affinity of the LNA nucleosides, allows the AIEX chromatography to be run at temperatures above 50 °C. In some embodiments the anion exchange high performance liquid chromatography (AIEX-HPLC) is performed at 60 °C or above. An increase in AIEX

chromatography temperature improves the separation of the duplex from the LNA probe significantly. The AIEX chromatography temperature should not exceed the melting temperature of the duplex between the oligonucleotide of interest and the LNA oligonucleotide probe. In some embodiments the AIEX chromatography is performed at a temperature between 60 °C and 2 °C below the melting temperature of the duplex between the oligonucleotide of interest and the LNA oligonucleotide probe

A major difference between a PNA probe and a LNA probe is the triplex formation of the PNA probe with DNA, LNA or RNA. PNA with poly pyrimidine segments (e.g. C and T nucleosides or variants thereof) form triplex with DNA, LNA or RNA oligonucleotides or polynucleotides containing poly purine sequences. The triplex formation makes PNA unreliable as probes for DNA, LNA or RNA oligonucleotides (such as oligonucleotides of interest) or polynucleotides with homo purine segments (see for example Knudsen H. et al. 1996, Nucleic Acid Research, Vol. 24 (3) pp. 494-500; Nielsen P. E et al 1999, Current Issues Molec. Biol Vol 1 (2) pp. 89-104; Hansen M. E et al Nucleic Acids Research, 2009, vol.37 (13) pp.4498-4507 and McNeer et al. 2013, Gene Therapy Vol. 20 pp. 658-669). PNA triplex formation can also be found where the PNA sequence is a mixed pyrimidine-purine sequence but has a surplus of pyrimidine. The stability and amount of triplex formed will then vary with the salt concentration and temperature and time (See for example Knudsen H. et al. 1996, Nucleic Acid Research, Vol. 24 (3) pp. 494- 500; Lesnik E. et al 1997, Nucleosides and Nucleotides, Vol. 16pp. 1775-1779 and Nielsen P. E et al, 1999, Current Issues Molec. Biol Vol. 1 (2) pp. 89-104). The formation of PNA triplexes are salt dependent and an anion exchange chromatogram with an increasing salt gradient is likely to result in less defined peaks or even two peaks, one with duplex PNA-DNA (LNA or RNA) and one with triplex PNA-DNA/LNA/RNA-PNA.

In one embodiment the LNA oligonucleotide probe comprises or consists of phosphodiester linkages. Generally the oligonucleotide of interest contains phosphorothioate linkages The phophodiester linkages in the LNA oligonucleotide probe have the advantage that it makes the probe smaller and less lipophilic than the phosphorothioate linked oligonucleotide of interest. . This leads to a very early elution in the AIEX chromatography of LNA oligonucleotide probes with phosphodiester linkages and serves to increase the separation of the LNA oligonucleotide probe from the oligonucleotide of interest. Therefore, a high excess of the LNA oligonucleotide probe can be used to kinetically control the hybridization process without establishing a step to extract the excess probe from the sample. The section "LNA oligonucleotide probe" below describes further details in relation to the construction of the LNA oligonucleotide probe.

Using a fluorescent label on the LNA oligonucleotide probe as opposed to an unlabeled LNA oligonucleotide probe detected by UV further serves to increase the high selectivity and sensitivity of the method of the present invention. The advantage of the fluorescent probe is that the signal intensity only depends on the fluorescence signal after the hybridization procedure and is independent of the UV extinction coefficients of the unlabeled probe and other biological background. As shown in the examples the use of a fluorescent label increases the detection limit at least 340 times, such as at least 400, 500, 750, 1000, 1500 times and such as at least 2000 times when compared to UV detection. The the oligonucleotide detection method of the invention can quantify the oligonucleotide concentration in the sample down to the nanomolar level (1 x10 ^~9 M), more preferably down to picomolar level (1 x10 ^"12 M) or most preferably down to 1 x10 ^-14 M.

Most of the previously described assays require individual calibration curves due to the variable unspecific background from different tissues or plasma. In contrast, unspecific background signals do not interfere with the assay of the present invention. Hence, in preferred

embodiments of the invention, calibration curves generated from a dilution series in buffer can be used for tissue and plasma samples.

In a further embodiment, a known concentration of the oligonucleotide of interest is added to the sample with an unknown concentration of the oligonucleotide of interest, and is also added to the calibration and blank sample. This is preferably done before addition of the LNA

oligonucleotide probe. This approach improves the sensitivity of the assay in that it identifies the exact elution time for the oligonucleotide of interest in the chromatogram, thereby improving the integration of the peak of interest.

The sections below contain detailed descriptions of the oligonucleotide of interest as well as the LNA oligonucleotide probe relating to the method of detection described above. LNA oligonucleotide probe

In order for the LNA oligonucleotide probe (also termed LNA probe) to detect the

oligonucleotide of interest with high sensitivity and reproducibility it is important that the probe is fully complementary to a stretch of the oligonucleotide of interest that is long enough to form a stable duplex that can be isolated by AIEX-HPLC.

The hybridization or duplex formation between the oligonucleotide of interest and the LNA oligonucleotide probe can be assessed by the Tm. In a preferred embodiment the Tm of the duplex between LNA oligonucleotide probe and the oligonucleotide of interest is above 60 °C.

The total length of the LNA oligonucleotide probe is generally defined by the length of the oligonucleotide of interest. In some embodiments the length of the LNA oligonucleotide probe is between 7 to 30 nucleotides in length, such as from 8 to 25, such as 9 to 22, such as from 10 to 18, such as from 1 1 to 16 or 12 to 14 contiguous nucleotides in length. In some embodiments, the LNA oligonucleotide probe comprises or consists of 7 or more nucleotides, such as 8 or more nucleotides, such as 9 or more nucleotides, such as 10 or more nucleotides, such as 1 1 , 12, 13 or 14 nucleotides. Preferably, the LNA oligonucleotide probe does not exceed the length of the oligonucleotide of interest by more than 5 nucleotides, such as by 4, 3, 2 or 1 nucleotides. In some embodiments the LNA oligonucleotide probe does not exceed the length of the oligonucleotide of interest. In some embodiments the LNA oligonucleotide probe is designed such that at least one LNA nucleoside in the LNA oligonucleotide probe forms a base pair upon duplex formation with a corresponding 2' sugar modified nucleoside in the oligonucleotide if interest. The 2' sugar modified nucleoside in the oligonucleotide of interest is preferably selected from high affinity nucleoside analogues, such as 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'- alkoxy-RNA, 2'-0-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides . Preferably, the corresponding 2' sugar modified

nucleoside in the oligonucleotide of interest is a LNA nucleotide resulting in the formation of an LNA:LNA base pair between the LNA oligonucleotide probe and the oligonucleotide of interest when a duplex between the two oligonucleotides is formed. Indeed, due to the very high stability of LNA:LNA duplexes (Fakhfakh et al, 2015, American inst. Of Chem. Engineers vol 61 , p.

271 1 ), the formation of such duplexes is considered to be particularly effective with respect to increasing the affinity between the LNA oligonucleotide probe and the oligonucleotide of interest. The ability to design LNA oligonucleotide probes with both DNA and LNA or other alternative sugar modified nucleosides allow for optimization and fine-tuning of the binding affinity of the LNA oligonucleotide probe to the oligonucleotide of interest. A duplex between the LNA oligonucleotide probe and the oligonucleotide of interest may therefore comprise different types of base pairs (the first being probe and the second the oligonucleotide of interest) such as 2' sugar modified:2' sugar modified (e.g. MOE:MOE, MOE:LNA, LNA:MOE, LNA:LNA, LNA:cET, cET:LNA, cET:cET etc); 2' sugar modified:DNA (e.g. MOE:DNA, LNA:DNA; cET:DNA etc.); DNA: 2' sugar modified (e.g. DNA:MOE, DNA:LNA; DNA:cET: etc.) or DNA:DNA base pairs. In some embodiments the LNA oligonucleotide probe does not contain more than 4 contiguous DNA nucleosides, more preferably the LNA oligonucleotide probe does not contain more than 3 contiguous DNA nucleosides.

In some embodiments the length of the complementary region between the LNA oligonucleotide probe and the oligonucleotide of interest comprises least 7 fully complementary contiguous nucleotides, such as at least 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 fully

complementary contiguous nucleotides. The longer the oligonucleotide of interest is the longer the complementary region may be. In some embodiments the LNA oligonucleotide probe comprises between 7 and 20 complementary nucleosides, such as between 8 and 18 complementary nucleosides, such as between 9 and 16 complementary nucleosides, such as between 10 and 14 complementary nucleosides, where the complementarity is to the oligonucleotide of interest.

In some embodiments at least 50% of the nucleotides in the oligonucleotide of interest form a sequence of contiguous nucleotides that are fully complementary to the LNA oligonucleotide probe with the proviso that the contiguous sequence is not shorter than 7 nucleotides (i.e. a duplex of at least 7 nucleotides can be formed between the oligonucleotide of interest and the LNA oligonucleotide probe). Preferably, at least 75%, 80%, 85%, 90% or 95% of the nucleotides in the oligonucleotide of interest form a sequence of contiguous nucleotides that are fully complementary to the LNA oligonucleotide probe with the condition that the contiguous sequence is not shorter than 10 or 12 nucleotides. Most preferably 100% of the oligonucleotide of interest is fully complementary to the LNA oligonucleotide probe. For the detection of metabolites of the oligonucleotide of interest the remaining part of contiguous sequence capable of forming a duplex with the LNA oligonucleotide probe may obviously be shorter than 7 nucleotides due to the metabolism of the oligonucleotide of interest.

The presence of at least one LNA nucleoside in the LNA oligonucleotide probe serves to increases the affinity of the probe towards the contiguous nucleotides in the oligonucleotide of interest which is complementary to the probe. This results in very stable duplexes between the LNA oligonucleotide probe and the oligonucleotide of interest. In preferred embodiments the LNA oligonucleotide probe comprises at least 2 LNA nucleosides, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 LNA nucleosides. In a further embodiment the LNA oligonucleotide probe comprises at least one LNA nucleoside in the 3' end, such as at least two or three LNA nucleosides in the 3' end of the LNA oligonucleotide probe. This serves to increase the nuclease resistance of the LNA oligonucleotide probe.

The LNA oligonucleotide probe can be defined by the structure (Do-3-L _{1 -} 3-Do-3-L _{1 -} 3-Do- ₃ )n, wherein L is an LNA nucleoside, D is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or 2' substituted nucleoside, n is between 1 and 3. Preferably the total length of the LNA

oligonucleotide probe is maintained within the ranges described above such as from 8 to 30 nucleotides in length.

In further embodiments the LNA oligonucleotide probe is defined by the structure (L ₁ . ₃ -Do-3-L ₁ . ₃ ) _n wherein L is an LNA nucleoside, D is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or 2' substituted nucleoside, n is between 1 and 4.

In some embodiments D independently is 1 or 2 DNA units. In further embodiments L is independently 1 or 2 LNA units.

In some embodiments the LNA oligonucleotide probe comprises at least 30% LNA nucleosides complementary to the oligonucleotide of interest, preferably at least 50 % and more preferably at least 75% LNA nucleosides complementary to the oligonucleotide of interest.

In some specific embodiments the LNA oligonucleotide probe has a design selected from D _1-2 -L- D1-2-L-D1-2-L-D1-2-L-D1-2-L-D1-2 or L2-D1-2-L1-2-L1-2-D1-2-L2.

Specific designs may be selected from D-D-L-D-D-L-D-D-L-D-D-L-D-D-L-D-D; D-D-L-D-D-L-D- D-L-D-D-L-D-D-L-D; D-L-D-D-L-D-D-L-D-D-L-D-D-L-D; D-D-L-D-L-D-D-L-D-D-L-D-D-L-D; D-D- L-D-L-D-L-D-D-L-D-D-L-D; D-D-L-D-D-L-D-D-L-D-L-D-L-D; D-D-L-D-L-D-L-D-L-D-D-L-D; D-D-L- D-L-D-L-D-L-D-L-D or D-L-D-L-D-L-D-L-D-L-D, where L is an LNA nucleoside and D is a deoxyribonucleic acid (DNA).

Other specific designs may be selected from L-L-D-D-L-L-D-D-L-L; L-L-D-L-L-D-L-L; L-L-D-L-L- L-D-L-L; L-L-D-L-L-L-L-D-L-L; L-L-D-D-L-L-L-L-D-L-L or L-L-D-D-L-L-L-L-D-D-L-L, where L is an LNA nucleoside and D is a deoxyribonucleic acid (DNA).

In some embodiments the LNA oligonucleotide probe is constituted of 100% LNA nucleosides. LNA oligonucleotide probes with 100% LNA oligonucleotides may have a tendency to self- anneal if they are too long. The skilled person will know how to assess this. In the event where the LNA oligonucleotide probe is constituted of 100% LNA oligonucleotides it is between 6 and 12 nucleotides long, such as between 7 and 1 1 nucleotides long, such as between 8 and 10 nucleotides long. In some specific embodiments the LNA oligonucleotide probe has a design selected from L _6- i ₂ . In some embodiments, the LNA oligonucleotide probe comprises more than 60% pyrimidine nucleosides, such as C and/or T or variants thereof, such as more than 70 %, 80% or 90% pyrimidine nucleosides such as C and/or T or variants thereof.

In some embodiments the LNA oligonucleotide probe comprises or consists of phosphodiester linkages. In some embodiments at least 50% of the internucleoside linkages in LNA

oligonucleotide probe are phosphodiester linkages, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the LNA oligonucleotide probe are phosphodiester linkages. In some embodiments all of the

internucleoside linkages of the LNA oligonucleotide probe are phosphodiester linkages.

LNA oligonucleotide probe of the present invention is labelled with a fluorescent label. There are many such labels available to the skilled person. The following is a non-limiting list of suitable fluorescent labels which can be selected from fluorescein or variants thereof such as fluorescein phosphoramidite (FAM), 6-carboxyfluorescein (6-FAM), fluoresceine isothiocyanate (FITC), 6- carboxy-4',5'-dichloro-2',7'-dimethoxyfluoresceine (JOE), hexachloro-fluoresceine (Hex) and 5- Tetrachloro-Fluorescein (TET), or from rhodamine or variants thereof such as, Rhodamine

Green and carboxy-tetramethyl-rhodamine (TAMRA) or from cyanine or variants thereof such as Cy5, Cy55, Cy33 and Cy3, or from other florescent labels like Alexa Flour 488, ATTO 425, ATTO 465, ATTO 488 Redmond red and Yakima yellow. Other fluorescence labels known to a person skilled in the art can be used in the method. In some embodiments the fluorescent label is attached to the 5' end of the LNA oligonucleotide probe. When the 5' end is labelled the 3' end of the LNA oligonucleotide probe preferably contains at least one LNA nucleoside. In some embodiments the fluorescent label is attached to the 3' end of the LNA oligonucleotide probe. When the 3' end is labelled the 5' end of the LNA oligonucleotide probe preferably contains at least one LNA nucleoside.

The present invention also encompasses a fluorescently labelled LNA oligonucleotide probe as described above for use in a quantitative or qualitative analysis of a oligonucleotide of interest.

Oligonucleotide of interest

The oligonucleotides of interest suitable for detection with the method of the present invention are capable of modulating expression of a target nucleic acid or a target molecule. The modulation may achieved by hybridizing of the oligonucleotide of interest to the target nucleic acid or by binding of the oligonucleotide of interest the target molecule. The oligonucleotide of interest is a therapeutic oligonucleotide suitable for treatment or prophylaxis of a disease.

The oligonucleotide of interest to be qualitatively or quantitatively detected by the method of the invention can be selected from the group consisting of antisense oligonucleotides, siRNA, aptamers and spiegelmers or a mixture of such oligonucleotides. In some embodiments the antisense oligonucleotide of interest is capable of modulating the expression or activity of the target molecule by inhibiting or down-regulating it. Preferably, such modulation produces an inhibition of expression or activity of at least 20% compared to the normal expression level of the target, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% inhibition compared to the normal expression level or activity of the target molecule.

In one embodiment the target modulation can be triggered by the binding to non-nucleic acid molecule such as a protein, peptide, lipid or carbohydrate. In some embodiments the

oligonucleotide of interest is an aptamer or spiegelmer.

In another embodiment the target modulation can be triggered by the hybridization between a contiguous nucleotide sequence of the oligonucleotide of interest and the target nucleic acid. The target nucleic acid can for example be a RNA or DNA, such as micro RNA, non-coding RNA, viral RNA/DNA or a messenger RNA, such as a mature mRNA or a pre-mRNA.

In some embodiments the oligonucleotide of interest is a therapeutic oligonucleotide

oligonucleotide of 8 to 30 nucleotides in length. In some embodiments the oligonucleotide of interest is an antisense oligonucleotide or a siRNA. In some embodiments the oligonucleotide of interest is single stranded, and the probe complementary to the oligonucleotide of interest is also single stranded. In a preferred embodiment the oligonucleotide of interest is an antisense oligonucleotide. The antisense oligonucleotide comprises a contiguous nucleotide sequence of 8 to 30 nucleotides in length with at least 90% complementary to a target nucleic acid.

In some embodiments, the oligonucleotide comprises a contiguous sequence which is at least 90% complementary, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence thereof comprises or consists of 22 or less nucleotides, such as 20 or less nucleotides, such as 18 or less nucleotides, such as 14, 15, 16 or 17 nucleotides.

In some embodiments, the contiguous nucleotide sequence of the oligonucleotide of interest comprises or consists of 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length. In some embodiments, the contiguous nucleotide sequence of the oligonucleotide of interest comprises or consists of between 8 and 30 nucleosides, such as between 10 and 25 nucleosides, such as between 1 1 and 22

nucleosides, such as between 12 and 20 nucleosides, such as between 13 and 18 nucleosides, such as between 14 and 16 nucleosides. Incorporation of modified nucleosides into the oligonucleotide of interest may enhance the affinity of the oligonucleotide for the target nucleic acid. In that case, the modified nucleosides can be referred to as affinity enhancing modified nucleotides.

In an embodiment, the oligonucleotide of interest comprises comprises one or more sugar modified nucleosides, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 modified nucleosides. In an embodiment the oligonucleotide of interest comprises from 1 to 16 modified nucleosides, such as from 2 to 14 modified nucleosides, such as from 3 to 12 modified nucleosides, such as from 4 to 8 modified nucleosides, such as 5, 6 or 7 modified nucleosides. In an embodiment all the nucleosides of the oligonucleotide are modified nucleosides

In an embodiment, the oligonucleotide of interest may comprise modifications, which are independently selected from modified sugar and modified internucleoside linkage or a combination thereof. Preferably, the oligonucleotide comprises one or more sugar modified nucleosides, such as one or more 2' sugar modified nucleoside independently selected from the group consisting of 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'-alkoxy-RNA, 2'-0-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides.

In some embodiments, one or more sugar modified nucleoside is a locked nucleic acid (LNA) nucleoside, such as at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 of the modified nucleosides are LNA. In one embodiment the oligonucleotide of interest comprises between 2 and 14, such as between 3 and 12, 4 and 8, such as 5, 6 or 7 LNA nucleosides. In a still further embodiment all the modified nucleosides are LNA.

In some embodiments, the oligonucleotide of the interest comprises at least one LNA unit and at least one 2' substituted modified nucleoside.

In some embodiments, the oligonucleotide of interest comprises more than 60% purine nucleosides such as A and/or G or variants thereof, such as more than 70%, 80% or 90% purine nucleosides such as A and/or G or variants thereof.

In a further embodiment the internucleoside linkages in oligonucleotide of interest comprises or consists of phosphorothioate linkages. In some embodiments the oligonucleotide of interest comprises at least one modified internucleoside linkage. In a preferred embodiment the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages. In a preferred embodiment all the internucleotide linkages in the contiguous sequence of the oligonucleotide of interest are phosphorothioate linkages. The structural design of the oligonucleotide of interest may be selected from gapmers, mixmers, headmers, tailmers and totalmers. For reference to these designs see Antisense Drug

Technology, Principles, Strategies, and Applications, 2 ^nd edition, chapter 19.6.1 .

In a further embodiment the oligonucleotide of interest is covalently attached to at least one conjugate moiety.

Use of the methods of the invention

The method of the invention will be useful in quantitative or qualitative analysis of the metabolism of a compound comprising the oligonucleotide of interest. In particular the metabolism of compounds that constitute oligonucleotide conjugates. The present method will allow quantification of the cleavage of the oligonucleotide conjugate into free oligonucleotide and conjugate moiety either in vivo or in vitro. The method can also establish where such cleavage occur in vivo by analysing for example serum samples and target tissues such as adipose tissue, bone marrow, brain, cartilage, cancer cells, heart, immune cells, intestine, kidney, liver, lung, muscle, neurons, pancreatic cells, etc.

The method may also be used to detect the metabolism of the oligonucleotide of interest into oligonucleotide fragments. Elution depends strongly on the metabolite length, the shorter the metabolite, the earlier it elutes from the HPLC column within the gradient.

In one embodiment, the method is used for quantitative and qualitative detection of an antisense oligonucleotide and derivatives. In yet another embodiment the method can be used for the quantitative and qualitative detection of the in vivo metabolism of a therapeutic or diagnostic antisense oligonucleotide.

In another preferred embodiment, the method is used for quantitative and qualitative detection of siRNA and derivatives. In yet another embodiment the method can be used for the quantitative and qualitative detection of the in vivo metabolism of therapeutic or diagnostic siRNA.

In one embodiment the method is used for quantitative and qualitative detection of an antisense oligonucleotide of interest from an in vitro cell culture that have been exposed to the

oligonucleotide of interest. The exposure can for example be via gymnosis or transfection. In this case the sample is obtained from the in vitro cell culture.

In another embodiment the method is used for quantitative and qualitative detection of a siRNA of interest from in vitro cell cultures that have been exposed to the siRNA of interest. The exposure can for example be transfection. In this case the sample is obtained from the in vitro cell culture. In yet another embodiment the method is used for quantitative and qualitative detection of aptamers. Preferably, said aptamers are spiegelmers with L-ribose (L-RNA) or L-deoxyribose (L-DNA). In one embodiment, said aptamer is pegylated.

In yet another embodiment the method can be used in the quantitative or qualitative analysis of intracellular uptake of the oligonucleotide of interest delivered in vitro or in vivo.

In yet another embodiment the method can be used in a quantitative or qualitative analysis of the tissue distribution of the oligonucleotide of interest delivered in vivo. The analysis will be performed on samples isolated from the animal at one or more given time points after the in vivo administration to an animal of the oligonucleotide of interest. Relevant samples are for example blood, plasma, urine or tissue.

Kits

This invention is also directed to kits suitable for performing an assay which detects the presence, absence or numbers of oligonucleotide molecules of interest and potentially its metabolites in a sample. The kits of this invention comprise a ready-to-use plate preparation comprising one or more LNA oligonucleotide probes and all other reagents or compositions necessary to perform the assay.

One embodiment is a kit for use in qualitative and quantitative detection of a oligonucleotide of interest as defined in the section "oligonucleotide of interest", comprising a plate preparation with a fluorescently labelled LNA oligonucleotide probe as defined in the section "LNA oligonucleotide probe" and optionally a protease. The use of the kit simplifies the performance of the assay and improves the reproducibility of the assay. Preferred kits of the invention make use of a fully automated robotic system for oligonucleotide detection, where all reagents are added by a pipetting robot. Thus the reproducibility of the assay is further improved. In addition, this setup can be used for high-throughput analysis of oligonucleotides in different samples. In one preferred embodiment, the kits comprise a 96 well- plate preparation, in yet another embodiment the kits comprise a 384 well plate preparation.

EMBODIMENTS

1 . A method for detecting an oligonucleotide of interest, comprising the steps of

a) obtaining a sample containing or suspected of containing said oligonucleotide of interest;

b) forming a hybridization mixture by contacting the sample with a fluorescently labelled LNA oligonucleotide probe which is fully complementary to at least 7 or more contiguous nucleotides of said oligonucleotide of interest; c) separating hybridized moieties formed between said oligonucleotide of interest and said LNA oligonucleotide probe from unhybridized moieties by anion exchange high performance liquid chromatography (AIEX-HPLC); and

d) qualitatively or quantitatively detecting said hybridized moieties by fluorescence spectroscopy.

2. The method according to embodiment 1 , wherein the oligonucleotide of interest is a therapeutic oligonucleotide.

3. The method according to any one of embodiments 1 or 2, wherein the oligonucleotide of interest is of 8 to 30 nucleotides in length.

4. The method according to any one of embodiments 1 to 3, wherein the oligonucleotide of interest is selected from the group consisting of antisense oligonucleotides, siRNA, aptamers and spiegelmers or a mixture of such oligonucleotides.

5. The method according to embodiment 4, wherein the oligonucleotide of interest is an antisense oligonucleotide.

6. The method according to any one of embodiments 1 to 5, wherein the oligonucleotide of interest and the oligonucleotide probe are single stranded.

7. The method according to any one of embodiments 1 to 6, wherein the oligonucleotide of interest comprises one or more sugar modified nucleosides.

8. The method according to embodiment 7, wherein the one or more sugar modified nucleoside is a 2' sugar modified nucleoside independently selected from the group consisting of 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'-alkoxy-RNA, 2'-0-methoxyethyl-RNA, 2'-amino-DNA, 2'- fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides.

9. The method according to embodiment 7 or 8, wherein the one or more sugar modified nucleoside is a LNA nucleoside.

10. The method according to any one of embodiments 1 to 9, wherein the oligonucleotide of interest comprises between 2 and 14 LNA nucleosides.

1 1 . The method according to any one of embodiments 1 to 10, wherein the oligonucleotide of interest is a gapmer or a mixmer.

12. The method according to embodiment 1 1 , wherein the gapmer is a mixed wing gapmer. 13. The method according to any one of embodiments 1 to 12, wherein the oligonucleotide of interest comprises more than 60% purine nucleosides.

14. The method according to any one of embodiments 1 to 13, wherein the internucleoside linkages in the oligonucleotide of interest comprises or consists of phosphorothioate linkages. 15. The method according to any one of embodiments 1 to 14, wherein the oligonucleotide of interest is covalently attached to at least one conjugate moiety.

16. The method according to any one of embodiments 1 to 15, wherein the LNA

oligonucleotide probe comprises at least 30% LNA nucleosides complementary to the oligonucleotide of interest, preferably at least 50 % and more preferably at least 75% LNA nucleosides complementary to the oligonucleotide of interest.

17. The method according to any one of embodiments 1 to 16, wherein the LNA

oligonucleotide probe upon duplex formation with the oligonucleotide of interest forms at least one 2' sugar modified nucleoside: 2' sugar modified nucleoside base pair with the

oligonucleotide of interest.

18. The method according to claim 17, wherein the LNA oligonucleotide probe upon duplex formation with the oligonucleotide of interest forms at least one LNA:LNA base pair.

19. The method according to any one of embodiments 1 to 18, wherein the LNA

oligonucleotide probe has the structure (Do-3-L _{1 -} 3-Do-3-L _{1 -} 3-Do- ₃ )n, wherein L is an LNA nucleoside, D is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or 2' substituted nucleoside, n is between 1 and 3.

20. The method according to any one of embodiments 1 to 16, wherein the LNA

oligonucleotide probe has the structure (L ₁ . ₃ -Do-3-L ₁ . ₃ )n wherein L is an LNA nucleoside, D is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or 2' substituted nucleoside, n is between 1 and 4.

21 . The method according to embodiment 19 or 20, wherein D independently is 0 to 2 DNA units.

22. The method according to embodiment 16 or 19, wherein the LNA oligonucleotide probe is constituted of 100% LNA nucleosides.

23. The method according to any one of embodiments 1 to 22wherein the LNA oligonucleotide probe comprises more than 60% pyrimidine nucleosides.

24. The method according to any one of embodiments 1 to 23, wherein the internucleoside linkages in the LNA oligonucleotide probe comprises or consists of phosphodiester linkages.

25. The method according to embodiment 24, wherein all the internucleoside linkages in the part of the LNA oligonucleotide probe which is complementary to the oligonucleotide of interest are phosphodiester linkages.

26. The method according to any one of embodiments 1 to 25, wherein the LNA

oligonucleotide probe is labelled with a fluorescent label is selected from fluorescein or variants thereof such as fluorescein phosphoramidite (FAM), 6-carboxyfluorescein (6-FAM), fluoresceine isothiocyanate (FITC), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluoresceine (JOE), hexachloro- fluoresceine (Hex) and 5-Tetrachloro-Fluorescein (TET), or from rhodamine or variants thereof such as, Rhodamine Green and carboxy-tetramethyl-rhodamine (TAMRA) or from cyanine or variants thereof such as Cy5, Cy55, Cy33 and Cy3, or from other florescent labels like Alexa Flour 488, ATTO 425, ATTO 465, ATTO 488 Redmond red and Yakima yellow.

27. The method according to embodiment 26, wherein the fluorescent label is selected from fluorescein or variants thereof such as fluorescein phosphoramidite (FAM), 6-carboxyfluorescein (6-FAM), hexachloro-fluoresceine (Hex) and 5-Tetrachloro-Fluorescein (TET).

28. The method according to any one of embodiments 1 to 27, wherein the anion exchange high performance liquid chromatography (alEX-HPLC) is performed at 60 °C.

29. The method according to any one of embodiments 1 to 27, for use in a quantitative or qualitative analysis of the in vivo or in vitro metabolism of a compound comprising the oligonucleotide of interest.

30. The method according to embodiment 29, wherein the compound is an oligonucleotide conjugate, and the metabolism detected is the cleavage of the conjugate moiety from the oligonucleotide of interest.

31 . The method according to any one of embodiments 1 to 26, for use in a quantitative or qualitative analysis of the intracellular uptake of the oligonucleotide of interest in vitro or in vivo.

32. The method according to embodiment 31 , wherein the uptake is analysed in a liver and/or kidney sample.

33. The method according to any one of embodiments 1 to 31 , wherein the sample is obtained from an in vitro cell culture.

34. The method according to any one of embodiments 1 to 29 for use in a quantitative or qualitative analysis of the tissue distribution of the oligonucleotide of interest delivered in vivo. 35. The method according to any one of embodiments 1 to 34, wherein the sample is blood, plasma, urine or tissue isolated from an animal following in vivo administration of the

oligonucleotide of interest.

36. The method according to embodiment 34 or 35, wherein the tissue is selected from the group consisting of adipose tissue, bone marrow, brain, cartilage, cancer cells, heart, immune cells, intestine, kidney, liver, lung, muscle, neurons and pancreatic cells.

37. The method according to any one of embodiments 1 to 36, wherein the sample is treated with protease prior to addition of the LNA oligonucleotide probe or in connection with the addition of the LNA oligonucleotide probe. 38. The method according to any one of embodiments 1 to 37, wherein a known concentration of the oligonucleotide of interest that should be detected is added to the sample before contacting the sample with the fluorescently labelled LNA oligonucleotide probe.

39. A fluorescently labelled LNA oligonucleotide probe for use in a quantitative or qualitative analysis of a oligonucleotide of interest, wherein the LNA oligonucleotide probe is defined in any one of embodiments 1 or 16 to 26.

40. A kit for use in qualitative and quantitative detection of an oligonucleotide of interest as defined in any one of embodiments 6 to 14, comprising a plate preparation with a fluorescently labelled LNA oligonucleotide probe as defined in any one of embodiments 1 or 16 to 26.

41 . The kit according to embodiment 40, further comprising a protease.

42. The kit according to embodiment 40 or 41 , wherein the plate preparation is a 96 well or 384 well plate preparation.

EXAMPLES Materials and Methods Target oligonucleotides:

where capital letters indicate beta-D-oxy LNA nucleosides and lower case letters indicate DNA nucleosides. LNA cytosines were 5-methyl cytosine. Internucleoside linkages in the

oligonucleotide sequence were all phosphorothioate internucleoside linkages.

The target oligonucleotides were essentially synthesized as described in the LNA

oligonucleotide synthesis section below.

Probe oligonucleotides:

nucleosides; italic, underlined letters indicate PNA nucleosides; lower case letters indicate DNA nucleosides. LNA cytosines were 5-methyl cytosine. Internucleoside linkages in the LNA probes are phosphodiester linkages.

The duplex formed between the target oligonucleotide and the LNA/PNA oligonucleotide probes are shown below.

5 ¹ GTtgacactgTC 3 ¹

3 ' CAACTGTGACAG 5'

3' caactgtgacag 5 '

5 ^f CcAttGTcaCaCtCC 3 '

3' ggTaaCagTgtGagGt 5'

3' ggtaacagtgtgaggt 5 '

The LNA probes were essentially synthesized as described in the LNA oligonucleotide synthesis section below. The PNA probes were purchased from PNA Bio. LNA Oligonucleotide synthesis

LNA Oligonucleotide synthesis is generally known in the art. Below is a protocol which may be applied. The oligonucleotides of the present invention may have been produced by slightly varying methods in terms of apparatus, support and concentrations used.

LNA Oligonucleotides are synthesized on uridine universal supports using the phosphoramidite approach on an Oligomaker 48 at 1 μηιοΙ scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia for 5-16hours at 60 ^° C. The oligonucleotides are purified by reverse phase HPLC (RP-HPLC) or by solid phase extractions and characterized by UPLC, and the molecular mass is further confirmed by ESI-MS.

Elongation of the oligonucleotide:

The coupling of β-cyanoethyl- phosphoramidites (DNA-A(Bz), DNA- G(ibu), DNA- C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA- G(dmf), or LNA-T) is performed by using a solution of 0.1 M of the 5'-0-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. For the final cycle a phosphoramidite with desired

modifications can be used, e.g. a C6 linker or a hexaethylene glycol linker for attaching a conjugate group such as a fluorescein. Thiolation for introduction of phosphorthioate linkages is carried out by using xanthane hydride (0.01 M in acetonitrile/pyridine 9:1 ). Phosphordiester linkages can be introduced using 0.02 M iodine in THF/Pyridine/water 7:2:1 . The rest of the reagents are the ones typically used for oligonucleotide synthesis.

For post solid phase synthesis conjugation a commercially available C6 aminolinker

phorphoramidite can be used in the last cycle of the solid phase synthesis and after

deprotection and cleavage from the solid support the aminolinked deprotected oligonucleotide is isolated. The conjugates are introduced via activation of the functional group using standard synthesis methods.

Purification by RP-HPLC:

The crude compounds are purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150x10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile is used as buffers at a flow rate of 5 mL/min. The collected fractions are lyophilized to give the purified compound typically as a white solid.

Abbreviations:

DCI:4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4'-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

HPLC Eluation Protocol:

The quantification of the target oligonucleotide was done using a DIONEX Ultimate 3000 HPLC system equipped with a fluorescence detector or an UV detector. The column used was an AEI column (DNA pac 100 2x250, Thermo Scientific) heated to 60 °C.

The elution buffers used were:

Buffer A (10 mM NaCI04, 1 mM EDTA, 20 mM TRIS-HCL pH 7.8) and

Buffer B (1 mM NaCI04, 1 mM EDTA, 20 mM TRIS-HCL pH 7.8).

The elution gradient applied was

0 min. 0.25 mL/min. 100 % buffer A,

12 min. 0.25 mL/min. 75% Buffer A and 25 % buffer B,

22 min. 0.25 mL/min. 45% Buffer A and 55 % buffer B,

25 min. 0.25 mL/min. 100 % B,

30 min. 0. 25 mL/min. 100 % buffer A,

31 min. 0. 5 mL/min. 100 % buffer A,

33 min. 0. 5 mL/min. 100 % buffer A,

35 min. 0. 25 mL/min. 100 % buffer A

40 min. 0. 25 mL/min. 100 % buffer A.

The fluorescence was measured by emission at 518 nm after exitation at 494 nm and the UV was measured by emission at 260 nm. Example 1 : Detection of target oligonucleotide 1 in Monkey serum using LNA probe

The purpose of the present experiment is to establish the quantization level of the target LNA antisense oligonucleotide 1 (AS01 ) using either the standard UV detection method or a fluorescently labelled LNA oligonucleotide probe composed of 100% LNA and a fluorescence detector. Plasma from cynomolgus monkeys was spiked with LNA antisense oligonucleotide 1 to a concentration of 10000 ng/mL (2.392 nmol/mL). A dilution series of the sample was made by diluting to half the concentration 12 times with plasma to an end concentration of 2.44 ng/mL (0.584 pmol/mL). A similar dilution series was made for the UV detection method, starting at a concentration of 13333 ng/mL. The dilution series is shown in Table 1 below.

All the samples were treated over night with protease k (Sigma P4850) to a concentration of 1 mg/mL at 37 °C. The samples can be used as they are or diluted with buffer A and then added in 2-5 molar equivalents of the fluorescently labelled LNA oligonucleotide probe. In this example 5 nmol/mL of the fluorescently labelled LNA oligonucleotide probe 1 (LNA-P1 ) was added. Nothing was added to the samples for UV detection. All the samples were heated to 60 °C for 2 minutes and then allowed to return to room temperature.

From each of the samples 50 μ\- was injected into a DIONEX Ultimate 3000 HPLC system equipped with an UV detector or a fluorescence detector. The elution was performed as described in the Materials and Methods section.

The results are shown in Table 1 .

Table 1 : Quantization of AS01 by fluorescence or UV detection.

Fluorescence detection UV detection

AS01 concentration Emmision area AS01 concentration Uv absorbtion area ng/mL pmol/mL 518 nm ng/mL pmol/mL 260 nm

4.88 1 .17 317.6

2.44 0.58 341 .4

n.d.= not determined

The slope of regression and the correlation of the data points in Table 1 are shown in Figure 1 A. Figure 1 B shows the slope of regression and the correlation of the data points in the area indicated with a black square in Figure 1 A. In the lower concentration area there is a linear regression between the points down to 4.88 ng/mL. The lowest quantitation level of LNA antisense oligonucleotide 1 when spiked into monkey plasma was found to be 4.88 ng/mL or 1 .17 pmol/mL.

The lowest level of quantization for a LNA ASO using uv detection at 260 nm and the same AIEX HPLC conditions as in experiment 1 is 1667 ng/mL or 399 pmol/ml. In the present example the fluorescence hybridization assay lowered the detection and quantization levels with a factor of 340 times.

The chromatograms for the quantization of AS01 at selected concentrations using fluorescence detection with LNA-P1 (chromatogram A-C) or UV detection (chromatogram D-F) are shown in Figure 2. The chromatograms for the fluorescence detection were run as repeats of the experiment in Table 1 , with the change that the concentrations of AS01 were identical to those used for the UV detection. In chromatogram E and F (UV detection) it can be seen that the peak containing AS01 has almost or totally disappeared indicating that the detection limit using the UV detection method has been reached. Using the fluorescence detection method of the invention (chromatogram A-C) the AS01 containing peak is well separated from the peak containing the LNA oligonucleotide probe (the peak extending out of the chromatogram), and the AS01 concentration can be quantified in all the indicated chromatograms.

Example 2 - Detection of target oligonucleotide 2 in Monkey serum using LNA probe

The purpose of the present experiment is to establish the quantization level of the target LNA antisense oligonucleotide 2 (AS02) using either the standard UV detection method or a fluorescently labelled LNA oligonucleotide probe composed of a mixture of LNA and DNA and a fluorescence detector.

Plasma from cynomolgus monkeys was spiked with LNA antisense oligonucleotide 2 to a concentration of 500 ng/mL (95 pmol/mL). A dilution series of the sample was made by diluting to half the concentration 1 1 times with plasma to an end concentration of 0.244 ng/mL (0.046 pmol/mL). A similar dilution series was made for the UV detection method, starting at a concentration of 20,000 ng/mL. The dilution series is shown in Table 2 below. All the samples were treated over night with protease k at 37 °C. To the samples to be detected by the fluorescence method of the present invention 1 .05 nmol/mL of the fluorescently labelled LNA oligonucleotide probe 2 (LNA-P2) was added. Nothing was added to the samples for UV detection. All the samples were heated to 60 °C for 2 minutes and then allowed to return to room temperature.

From each of the samples 50 μΙ_ was injected into a DIONEX Ultimate 3000 HPLC system equipped with an UV detector or a fluorescence detector. The eluation was performed as described in the Materials and Methods section.

The results are shown in Table 2.

Table 2: Quantization of AS02 by fluorescence or UV detection

n.d.= not determined

The slope of regression and the correlation of the data points in Table 2 are shown in Figure 3A. Figure 3B shows the slope of regression and the correlation of the data points in the area indicated with a black square in Figure 3A. The lowest quantitation level of LNA antisense oligonucleotide 2 when spiked into monkey plasma was found to be 3.91 ng/mL or 0.75 pmol/mL.

The lowest level of quantization for a LNA ASO using UV detection at 260 nm and the same AIEX HPLC conditions as in experiment 1 is around 1250 ng/mL. In the present example the fluorescence hybridization assay lowered the detection and quantization levels with a factor of about 320 times. Chromatograms for the quantization of AS02 at selected concentrations using fluorescence detection with LNA-P2 (chromatogram A-C) or UV detection (chromatogram D-F) are shown in Figure 4. In chromatogram E and F (UV detection) it can be seen that the peak containing AS02 has almost or totally disappeared indicating that the detection limit using the UV detection method has been reached. Using the fluorescence detection method of the invention

(chromatogram A-C) the AS02 containing peak is well separated from the peak containing the LNA oligonucleotide probe (the peak extending out of the chromatogram) at concentrations that are significantly lower than those used for the UV detection, and the AS02 concentration can be quantified in all the indicated chromatograms. Example 3: Detection of target oligonucleotide 1 in Monkey serum using PNA probe

The purpose of the present experiment is to establish the quantization level of the target LNA antisense oligonucleotide 1 (AS01 ) using the fluorescently labelled PNA oligonucleotide probe PNA-P1 .

The experiment was essentially conducted as described for LNA-P1 in Example 1 , exchanging the LNA-P1 probe with the PNA-P1 probe.

The results are shown in Table 3.

Table 3: Quantization of AS01 by fluorescence detection using a PNA probe.

n.d.= not determined

The slope of regression and the correlation of the data points in Table 3 are shown in Figure 5. There is a linear regression between the points down to 4.88 ng/mL. The LNA antisense oligonucleotide 1 when spiked into monkey plasma is detectable down to 2.44 ng/mL or 0.58 pmol/mL, however this is outside the linear regression and therefore the lowest quantifiable level using PNA-L1 probe is 4.88 ng/mL, which is similar to what was achieved with LNA-P1 in Example 1 .

Example 4 - Detection of target oligonucleotide 2 in Monkey serum using PNA probe

The purpose of the present experiment is to establish the quantization level of the target LNA antisense oligonucleotide 2 (AS02) using a fluorescently labelled PNA oligonucleotide probe PNA-P2.

The experiment was essentially conducted as described for LNA-P2 in Example 2, exchanging the LNA-P2 probe with the PNA-P2 probe. The PNA -P2 probe was not easily dissolved in water compared to the LNA-P2 probe.

The results are shown in Table 4.

Table 4: Quantization of AS02 by fluorescence detection using a PNA probe.

n.d.= not determined

The slope of regression and the correlation of the data points in Table 4 are shown in Figure 6. There is a linear regression between the points down to 7.81 ng/mL. The lowest quantitation level of LNA antisense oligonucleotide 2 when spiked into monkey plasma was found to be 7.81 ng/mL or 1 .48 pmol/mL, which is 2 fold higher than with the LNA-P2 probe in Example 2. The lower capabilities of the PNA probe probably stem from the reduced solubility. Often the solubility is related to the length of the PNA oligonucleotide. In these experiments the length of solvable PNA probe seems to be =< 16 bases.