The present invention relates to alkyne-LNA nucleotides, which can be efficiently derivatized by highly efficient copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry. In one embodiment, this allows preparation of fluorescent probes. In another embodiment, "click" chemistry allows preparation of peptide-oligonucleotide conjugates (POCs). Further derivatives of LNA nucleotides obtainable from "click" chemistry performed on alkyne-LNA nucleotides is also provided.

BACKGROUND TO THE INVENTION

Interactions of nucleic acids with biomolecules and small molecules are vital for living organisms, providing basis for cell division, growth and genetic heritance. However, in several autoimmune disorders, production of antibodies against an individual's own single- and double-stranded DNA occurs. If not diagnosed and treated early, autoimmune conditions can lead to serious health damage and even mortality. Autoimmune antibodies

(autoantibodies) against single-stranded DNA have been thoroughly studied by Glick et al. (see, Biochemistry 2001, 40, 2911-2922; and Biochemistry 2003, 42, 30-41) who showed that binding requires a sequence-specific hydrogen bonding between T and G bases of the 20-25-mer oligonucleotide region and the autoantibody. In turn, autoantibodies against double-stranded DNA, a hallmark of the important autoimmune conditions Antiphospholipide syndrome and Systemic lupus erythematosus (SLE), are known to be non-sequence-specific and have not been studied in detail.

Generally, monitoring interactions of nucleic acids by fluorescence is a convenient method for modern bioanalysis, which can be performed under native conditions without additional equipment or procedures by a simple change of color. Homogeneous (all-in-solution) fluorescence assays require use of fluorophores that are photostable and chemically stable and simultaneously provide high fluorescence quantum yields. Currently, fluorescent oligonucleotides containing bright cyanine and xanthene dyes are often applied in bioanalysis of nucleic acids and proteins, including antibodies. Moreover, the fluorescent probes have to bind target biomolecule with high affinity and specificity. Affinity-enhancing locked nucleic acids 2'-amino-LNA containing fluorescent polyaromatic hydrocarbons (PAHs) at 2'-amino group meet these requirements and are therefore very promising for diagnostics and research on diverse nucleic acid targets. Another appealing aspect of LNA/DNA probes is their very promising properties as aptamers in binding diverse protein targets.

The attractive biosensing properties of fluorescent probes labeled at 2'-position of uridine by highly efficient CuAAC reactions has been demonstrated (see Astakhova, I. K. ; Wengel, J. Chem. Eur. J. 2013, 19, 1112-1122.; Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.; Wolfbeis, O. S. ; Wagenknecht, H.-A. Bioconjugate Chem. 2009, 20, 558-564; and Rubner, M. M.; Holzhauser, C; Bohlander, P. R. ; Wagenknecht, H.-A. Chem. Eur. J. 2012, 18, 1299- 1302).

Other publications in the field include WO2013/0197794 and US2002/0086998.

However, obtaining fluorescent-labelled bioconjugates still remains a considerable challenge due to imperfection of current bioconjugation methods and limited availability of fluorescent molecules with appropriate functionality.

In addition to the above, synthetic peptide-oligonucleotide conjugates (POCs) are artificial tools of choice for detailed studies of protein- and peptide-nucleic acid interactions, as well as being promising bioconjugates for nucleic acid delivery and therapy. Novel POCs are described in which peptide chains are internally incorporated into oligonucleotides using a 2'- alkyne-2'-amino-LNA scaffold.

OBJECT OF THE INVENTION

It is an object of the invention to provide novel alkyne-LNA nucleotides, which can be readily derivatized by highly efficient copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry. A range of useful biological tools is thereby provided, which can be synthesized in a rapid and efficient manner.

In one embodiment, derivatization of said alkyne-LNA nucleotides with a fluorescent dye compound provides novel fluorescent probes. The resulting oligonucleotide probes efficiently bind complementary nucleic acids and human autoimmune antibodies against double- stranded DNA, in both cases being monitored by a bright fluorescence response. The fluorescence sensing approach is novel and simple since it allows efficient monitoring of diverse biomolecular interactions in solution following the same spectral principle and by a very simple fluorescence assay. Furthermore, incorporation of the fluorescent LNAs into the probes brings additional binding selectivity into the developed aptasensor. In another embodiment derivatization of said alkyne-LNA nucleotides with a peptide compound provides novel peptide-oligonucleotide conjugates (POCs) .

In other embodiments said alkyne-LNA nucleotides can be derived with a carbohydrate compound, a lipid compound or a protein compound.

SUMMARY OF THE INVENTION

The present invention provides an oligonucleotide comprising an alkyne-LNA nucleotide unit of formula (I) :

(I) wherein B is a purine or pyrimidine nucleobase; and A is selected from a single

bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

The invention also provides fluorescent LNA oligonucleotides, comprising a fluorescent-LNA nucleotide monomer of formula (II), as well as methods for their synthesis:

(Π) in which B is a purine or pyrimidine nucleobase; FL constitutes a fluorescent moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof. Peptide-oligonucleotide conjugates (POCs) of formula (III) are also provided :

(III)

In formula (III), B is a purine or pyrimidine nucleobase, preferably a pyrimidine nucleobase, more preferably thymine (T) . Q constitutes a peptide moiety. A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Cio alkyl, or combinations thereof.

A variety of other conjugates are also available via the "click" chemistry of the invention, as described in the following.

Further aspects of the invention will be understood from the following specification and the dependent claims.

LEGENDS TO THE FIGURES

Figures 1 and 2 show fluorescence detection of DNA/RNA (Figure 1 ) and monoclonal autoantibodies (Figure 2) in a medium salt buffer at 19 °C using 1.0 μΜ and 0.5 μΜ probes, respectively. In Figure 2, the probes in each case (in order from left to right for each target protein) are ssON7, ON7: DNA, ON7 : RNA, ssON8, ON8: DNA, ON8: RNA.

Figures 3A-3D show gel electrophoresis of 5'- ³² P-labelled oligonucleotides incubated with HS. DETAILED DISCLOSURE OF THE INVENTION

Locked nucleic acid (LNA) is a nucleotide analogue in which the sugar ring is constrained as part of a bicyclic system. This locks the furanose in the C3'-endo conformation, thus mimicking the conformation found in RNA. 2'-amino-LNA is an analogue of LNA, which has a higher affinity for complementary DNA/RNA strands than unmodified LNA.

2'-Amino-LNA

In a first embodiment, therefore, the invention provides an alkyne-LNA oligonucleotide. The oligonucleotide comprises an alkyne-LNA nucleotide unit of formula (I) :

(I)

In formula (I), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g. thymine (T) . Furthermore, A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Cio alkyl, or combinations thereof.

In all of the structures I-VI, A suitably consists of a combination of 2-5 of the above- mentioned groups. Suitably, A comprises -(C=0)-, and may form an amide with the N-atom of the LNA. Preferably, A is -(C=0)-(CH ₂ ) _n - (i .e. it forms a straight-chain alkyl amide linker with the N-atom of LNA) in which n = 1-5, such as 1 or 2, preferably 2.

In the present context, the term "Ci-Cio alkyl" is intended to mean a linear, cyclic or branched hydrocarbon group having 1 to 10 carbon atoms, respectively, such as methyl, ethyl, propyl, /so-propyl, cyclopropyl, butyl, /so-butyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl. Preferred examples of Ci_Ci ₀ alkyl as a part of the group A are Ci-C ₆ -alkyl, such as Ci-C ₄ -alkyl and C ₂ -C ₄ -alkyl, such as C ₂ alkyl.

In the present context, i. e. in connection with the term "Ci-Cio-alkyl", the term "optionally substituted" is intended to mean that the group in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from hydroxy, Ci- ₆ -alkoxy (I.e. Ci. ₅ -alkyl-oxy), amino, mono- and di(Ci_ ₅ -alkyl)amino, -N(Ci. ₄ -alkyl) ₃ ⁺ , cyano, nitro, Ci_ ₅ - alkylthio, and halogen . The term "halogen" includes fluoro, chloro, bromo, and iodo. In some important embodiments, Ci-Ci ₀ -alkyl (and any variants hereof) as a part of the group A is unsubstituted.

In the present context, the term "nucleobase" is intended to encompass purine and pyrimidine nucleobases, such as a pyrimidine nucleobases like thymine (T), uracil (U) and cytosine (C), and purine nucleobases like guanine (G) and adenine (A) .

Alkyne-LNA oligonucleotides in which B = thymine are indicated as M ¹ in this specification.

The alkyne-LNA oligonucleotide suitably comprises between 5-50 nucleotides, preferably between 5-25 nucleotides. At least one of these is the alkyne-LNA nucleotide monomer of formula (I) . It may be advantageous to include more than one, e.g. 2 or more, 3 or more, 5 or more alkyne-LNA nucleotide monomer of formula (I) in the alkyne-LNA oligonucleotides of the invention.

Particular alkyne-LNA oligonucleotides are those comprising monomer M ¹ , in which B is thymine, as shown in Schemes 1 and 2. Further details of the synthesis and characterisation of the alkyne-LNA nucleotide monomer with the structure M ¹ is to be found in the

experimental section.

Specific alkyne-LNA oligonucleotides are those listed in SEQ ID. 1-4. Especially interesting structural motif includes multiple incorporation (2-4) of alkyne groups separated by 2-5 nucleotides, such as those in SEQ ID. No. 2, 3 or 4.

Azide-alkyne cycloaddition using a copper (Cu) catalyst to form a triazole is known as the "click" reaction. This reaction exhibits high yield, good atom economy, high selectivity for terminal alkynes, and functions in vitro and in vivo.

The alkyne-LNA nucleotides of the invention can be readily derivatized by highly efficient copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry. Having a 2'- amino-LNA scaffold, the LNA-oligonucleotides disclosed herein demonstrate beneficial thermal stabilities of complementary complexes with DNA/RNA compared to those with a 2'-uridine scaffold .

Fluorescent Probes Fluorescent bioconjugates must be stable, easy to design and prepare, adaptable for both homogeneous and solid-phase detection and nano-assembly formats. They must also provide a robust and easily interpretable fluorescence signal.

2'-Amino-LNA analogues containing polyaromatic hydrocarbons (PAHs) meet these requirements. Fluorescence of PAH molecules (pyrene, various (phenylethynyl)pyrenes and perylene) is sensitive to even minor changes in their microenvironment by shifts of absorption/emission bands, and by formation of excited dimers, excimers and exciplexes. Another important advantage of using PAHs in molecular diagnostics and chemical biology studies is their high quantum yields and photostability.

The LNA skeleton, in turn, provides excellent selectivity and stability of binding DNA/RNA targets. CuAAC reaction was recently demonstrated as an ideal method for bioconjugation giving stable fluorescent 1,2,3-triazole products both in vitro and in vivo, and also for attachment of peptides to nucleic acids. Finally, spectral properties of PAHs can be remarkably improved by attaching to nucleotide via 1,2,3-triazole moiety.

An overview of this aspect of the invention is given in Scheme 1 :

Detection of autoimmune antibody

Alkyne-LNA monomer M Series of Fluorescent Hybridization and Homogeneous

Probes Fluorescence Assay

Scheme 1 : Modified monomer M ¹ for CuAAC preparation of the fluorescent probes targeting DNA/RNA and autoimmune antibodies.

The alkyne-LNA oligonucleotides of formula (I) can be labelled using the "click" reaction to form fluorescent LNA oligonucleotides. A method is therefore provided for synthesizing a fluorescent LNA oligonucleotide, said method comprising the step of reacting the

oligonucleotide set out above with a fluorescent dye compound having the structure:

FL-N ₃ in which FL constitutes a fluorescent moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the fluorescent dye compound having the structure FL-N ₃ so as to form a 1,2,3-triazole product.

Suitably, the fluorescent moiety FL is a cyanine, polyaromatic hydrocarbon (PAH, e.g .

perylene, pyrene), or xanthene fluorescent moiety. A suitable arrangement of linker atoms may be present between LF and N ₃ . The spectral properties of polyaromatic hydrocarbons can be remarkably improved by conjugation via the 1,2,3-triazole moiety.

The invention also relates to fluorescent LNA oligonucleotides per se. These comprise a fluorescent- LNA nucleotide monomer of formula (II) :

In formula (II), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) . FL constitutes a fluorescent moiety. Furthermore, A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof. In the fluorescent LNA oligonucleotides of Formula II, FL may comprise a cyanine, PAH (incl. perylene, pyrene) or xanthene fluorescent moiety. Particular alkyne-LNA oligonucleotides are those comprising monomers M ² -M ⁵ , in which B is thymine, and shown in Scheme 2. Further details of the synthesis and characterisation of alkyne-LNA

oligonucleotides with the structures M ² -M ⁵ is to be found in the experimental section.

Scheme 2: Incorporation of monomers M ^x -M ⁵ into synthetic oligonucleotides.

In view of the reagent for the preparation of Monomer M ¹ , it should be understood that the invention also provides an alkyne-LNA nucleoside of formula (X) :

wherein B is a purine or pyrimidine nucleobase; PI is H or a protecting group, e.g . DMT, MMT or Trityl (Tr) ; P2 is H, a protecting group or a coupling group, e.g . a phosphoramidite, e.g. -P(N(Pr) ₂ )OC ₂ H ₄ CN, a phosphodiester or phosphotriester, and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof. In a particular embodiment, the reagent is of the formula 3 above.

Particular fluorescent LNA oligonucleotides of the invention comprise a sequence with SEQ ID No. 5-20. SEQ ID No. 7, 8, 20 displayed the most promising sensing of DNA/RNA targets; SEQ ID No. 7 in complex with DNA showed high propensity in sensing autoimmune antibodies.

The present invention also relates to the duplex formed between fluorescent LNA

oligonucleotides of the invention and the complementary DNA/RNA strand.

The invention provides the use of the fluorescent LNA oligonucleotide according to the invention as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA. Further details of this are provided in the Examples and the discussion of Figure 1.

In addition, the present invention provides the use of the fluorescent LNA oligonucleotide according to the invention, or the duplex described above, for the detection of an

autoimmune antibody. Further details of this are provided in the Examples and the discussion of Figure 2.

Comparing the fluorescent probes disclosed herein to previously-reported LNA/DNA fluorescent probes (Astakhova et al, Chemistry, A European Journal, 2013, 19, 1112-1122) several advantages of the probes of the current invention are clear:

1. Convenient synthetic route, which allows rapid preparation of the fluorescent probes in high purity without additional purification steps. Moreover, the post-synthetic click chemistry approach allows simple and rapid screening of several fluorophores in context of fluorescence sensing of target DNA/RNA. This can be done using a library of fluorescent azides in the labeling step.

2. Having 2'-amino-LNA scaffold, the probes disclosed herein demonstrate beneficial thermal stabilities of complementary complexes with DNA/RNA compared to the probes having 2'- uridine scaffold, as described in the above reference (stabilization by +1.5 - +13.0 °C vs. destabilization by -1.5 - 6.5 °C, respectively).

3. Fluorescence quantum yields for the probes described in this invention are higher than previously described in the above reference (OF 0.54-1.00 vs. OF 0.07-0.20, respectively), resulting in superior fluorescence brightnesses (FB up to 80) of the former probes and, hence, in remarkably low limit of target detection values.

4. Fluorescence light-up upon binding complementary DNA/RNA is similar for 2'-amino-LNA and 2'-uridina probes and is up to 7.8-fold. However in case of the former probes (in the above reference) the light-up is observed for more dyes attached, while in the latter a 7.7- fold increase of the dyes' fluorescence was an exceptional case of only one FRET pair. The more consistent quenching of fluorescence in single-stranded LNA/DNA probes is caused by their increased propensity to form stable secondary, "duplex-like", structures (promoted by LNA monomers). Within the secondary structures, quenching of fluorescence of the attached hydrophobic dyes is more likely to occur.

Peptide-Oligonucleotide Conjugates (POCs)

Locked nucleic acids (LNA, 2'-amino-LNA and isomeric a-L-LNA analogues) display excellent biomedical properties within synthetic oligonucleotides, such as improved target binding affinity, selectivity and enzymatic stability.

The present invention describes novel peptide-oligonucleotide conjugates (POCs) in which peptide chains are internally incorporated into oligonucleotides using the 2'-alkyne- 2'- amino-LNA scaffold (Scheme 3). The structure of the alkyne-LNA nucleotides of the invention allows attachment of peptides using highly efficient post-synthetic CuAAC click chemistry.

Alkyne-Modified Internally Labeled POC; Duplex of POC with Target Oligonucleotide Single and Multiple Peptide DNA/RNA

Attachment

Scheme 3: General concept of CuAAC preparation of POCs

There are several advantages to this design. Firstly, selected internally-attached peptides have high potential to interact with oligonucleotide strands. Secondly, using highly efficient click chemistry, peptide chains can be incorporated into any position of oligonucleotide strand. Hence, the distance and orientation of the peptide residues can be readily controlled from well-known nucleic acid structure parameters. Thirdly, other peptides available as cell- penetrating and targeting specific cells can be covalently tethered to the 2'-alkyne.

The general structure of POCs designed in this study is shown in Scheme 3. First, 21mer oligonucleotides were synthesised containing single and double internal insertions of 2'- alkyne-LNA monomer M ¹ followed by CuAAC conjugation with two azide-functionalized peptide derivatives 6-7 (Scheme 4) . Combining bicyclic LNA skeleton and 2'-alkyne group, the monomer Mi is a very promising scaffold for preparation of POCs, providing the possibility of post-synthetic click chemistry along with precise positioning of the attached modifications within nucleic acid complexes, small to no duplex distortion and, hence, high target binding affinity.

Q = |-(CH ₂ ) ₄ -C-NH-KKKYGGFM (CH ₂ ) ₄ -C-NH-KKKYGGFL

Monomer M ⁶ Monomer M ⁷

QN ₃ = Azide 6 Azide 7

Scheme 4. Chemical structures of modified monomers M l and M6, M7, phosphoramidite reagent 3 and peptido-azides 6-7 used in this study. Sequences of natural enkephalins: YGGFM (Met-enkephalin) ; YGGFL (Leu-enkephalin) .

According to this embodiment, the invention therefore provides a method for synthesizing a peptide-oligonucleotide conjugate (POC), said method comprising the step of reacting the oligonucleotide according to the invention with a peptide compound having the structure:

Q-N ₃ in which Q constitutes a peptide moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the peptide compound having the structure Q-N ₃ so as to form a 1,2,3-triazole product. Suitably, one or two peptide moieties Q are reacted onto the alkylene-LNA oligonucleotide according to the invention. The invention also relates to peptide-oligonucleotide conjugates (POCs) per se. These comprise a peptide-LNA oligonucleotide monomer of formula (III) :

(III)

In formula (III), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g. thymine (T). Q constitutes a peptide moiety. A is selected from a single bond, -(C=0)-, - 0-, -NH-, -S- and optionally-substituted Ci-Cio alkyl, or combinations thereof.

Suitably, Q is a Met or Leu-enkephalin derivative containing additional lysine residues at the N-terminus. The most preferred POCs are those having the structures set out in SEQ ID. Nos. 21-26. Suitably, one or two peptide moieties Q are joined to said peptide-oligonucleotide conjugate (POC).

The invention also provides a duplex formed between the peptide-oligonucleotide conjugate (POC) of this embodiment, and the complementary DNA/RNA, as well as the use of the peptide-oligonucleotide conjugate (POC) according to this embodiment as a LNA/DNA probe for the detection of complementary DNA and/or RNA.

The peptide-oligonucleotide conjugate (POC) according to this embodiment can be used in therapy, in particular antisense therapy.

Other moieties

In addition to fluorescent moieties and peptides discussed above, "click" chemistry can also be used to attach other moieties to oligonucleotides by means of the alkyne-LNA nucleotides of the invention.

For instance, the oligonucleotide according to the invention can be reacted with a

carbohydrate compound having the structure:

CWO-N ₃ in which CHO constitutes a carbohydrate moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the carbohydrate compound having the structure CHO-N ₃ so as to form a 1,2,3-triazole product.

Therefore, carbohydrate-oligonucleotide conjugates (COCs) having the structure of Formula IV are provided :

(IV)

In formula (IV), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) . CHO constitutes a carbohydrate moiety. Furthermore, A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted C ₁ -C ₁ 0 alkyl, or combinations thereof. Suitable carbohydrate moieties CHO include monosaccharides and polysaccharides.

Similarly, the oligonucleotide according to the invention can be reacted with a lipid compound having the structure: in which L constitutes a lipid moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne- LNA nucleotide of formula (II) reacts with the azide moiety of the lipid compound having the structure L-N ₃ so as to form a 1,2,3-triazole product.

Therefore, Lipid-oligonucleotide conjugates (LOCs) having the structure of Formula V are provided :

In formula (V), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, thymine (T) . L constitutes a lipid moiety. Furthermore, A is selected from a single bond, (C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Cio alkyl, or combinations thereof.

Suitable lipid moieties L include C ₆ -C ₂ 4 fatty acid moieties.

Furthermore, the oligonucleotide according to the invention can be reacted with a proteii compound having the structure:

Pro-N ₃ in which Pro constitutes a protein moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the protein compound having the structure Pro-N ₃ so as to form a 1,2,3-triazole product.

Therefore, protein-oligonucleotide conjugates having the structure of Formula VI are provided :

(VI)

In formula (VI), B is a purine or pyrimidine nucleobase, such as a pyrimidine nucleobase, e.g . thymine (T) . Pro constitutes a protein moiety. Furthermore, A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof. EXAMPLES General

Reagents obtained from commercial suppliers were used as received. Phosphoramidite reagent 3 was prepared as described in Scheme 2. Fluorescent azides and TBTA for click chemistry were obtained from Lumiprobe LLC. 9,10-Diphenylantracene (DPA), perylene, crezyl violete perchlorate and oxazine 170 used in spectral studies were recrystallized. HPLC grade light petroleum ether, methanol, ethanol and DMF were distilled and stored over activated 4 A molecular sieves. DCM was always used freshly distilled over CaH ₂ . Other reagents and solvents were used as received. Photochemical studies were performed using spectroquality methanol, ethanol and cyclohexane.

Stock solutions for click chemistry were prepared as described. Click reactions were performed in 1.5 mL eppendorf tubes (monomers M ² -M ⁴ ) or 1 mL reactor tubes (monomer M ⁵ ) under argon using vigorous stirring in Emrys Creator (Personal Chemistry) in the latter case.

Oligonucleotide synthesis was carried out on a Perspective Biosystems Expedite 8909 instrument in 1 μιηοΙ scale using manufacturer's standard protocols. For incorporation of monomer M ¹ a hand-coupling procedure was applied (20 min coupling). The coupling efficiencies of standard DNA phosphoramidites and reagent 1 based on the absorbance of the dimethoxytrityl cation released after each coupling varied between 95% and 100%. Cleavage from solid support and removal of nucleobase protecting groups was performed using 32% aqueous ammonia and methylamine 1 : 1, v/v, for 4 h at rt. The resulting oligonucleotides were purified by DMT-ON RP-HPLC using the Waters System 600 equipped with Xterra MS C18-column (5 μιη, 150 mm x 7.8 mm). Elution was performed starting with an isocratic hold of A-buffer for 5 min followed by a linear gradient to 70% B-buffer over 40 min at a flow rate of 1.0 mL/min (A-buffer: 0.05 M triethyl ammonium acetate, pH 7.4; B-buffer: 25% buffer A, 75% CH ₃ CN). RP-purification was followed by detritylation (80% aq. AcOH, 30 min), precipitation (acetone, -18 °C, 12 h) and washing with acetone three times. The identity and purity of oligonucleotides was then verified by MALDI-TOF mass spectrometry and IC-HPLC, respectively. IC-HPLC was performed using the Merck Hitachi LaChrom instrument equipped with Dionex DNAPac Pa-100 column (250 mm x 4 mm). Elution was performed starting with an isocratic hold of A- and C-buffers for 2 min followed by a linear gradient to 60% B-buffer over 28 min at a flow rate of 1.0 mL/min (A-buffer: MQ water; B-buffer: 1M NaCI0 ₄ , C- buffer: 25mM Tris-CI, pH 8.0). MALDI-TOF mass-spectrometry analysis was performed using a MALDI-LIFT system on the Ultraflex II TOF/TOF instrument from Bruker and using HPA- matrix (10 mg 3-hydroxypicolinic acid, 50 mM ammonium citrate in 70% aqueous acetonitrile). Unmodified DNA/RNA strands were obtained from commercial suppliers and used without further purification.

Synthesis of modified monomer M ¹

See Scheme 2. Reagents and conditions: (i) Pent-4-ynoic acid, HATU, DIPEA, DMF, rt, 1 h, 71%; (ii) NC(CH ₂ ) ₂ OP(N(i-Pr)2)2, diisopropylammonium tetrazolide, DCM, rt, 24 h, 73%. DMT = 4,4 ^' -dimethoxytrityl.

(l/?,3/?,4/?,7S)-l-(4,4'-Dimethoxytrityloxymethyl)-7-hydr oxy-5-(pent-4-ynoyl)-3- (thymin-l-yl)-2-oxa-5-azabicyclo[2.2.1]heptane (2). Pent-4-ynoic acid (287 mg, 2.93 mmol) and HATU (1.11 g, 2.92 mmol) were dissolved in anhydrous DMF (12 mL). DIPEA (0.82 mL, 4.71 mmol) was added and the reaction mixture was stirred at room temperature for 10 min. A solution of nucleoside 1 (1.50 g, 2.62 mmol) in anhydrous DMF (12 mL) was added dropwise, and the reaction mixture was stirred at room temperature for a further 1 h. The reaction mixture was diluted with ethyl acetate (150 mL) and washed with a saturated aqueous solution of NaHC0 ₃ (2 x 475 mL) and water (6 x 50 mL). The aqueous phases were back-extracted in portions with ethyl acetate (160 mL in total). The combined organic phases were dried over Na ₂ S0 ₄ , filtered and the solvent removed in vacuo. The residue was purified by silica gel column chromatography (0 - 5% MeOH/CH ₂ CI ₂ ) to afford a rotameric mixture (~0.65:0.35 by ^X H NMR) of nucleoside 2 as an off-white foam (1.21 g, 71%). R _f 0.57 (10% MeOH/CH ₂ CI ₂ ); ¹ H NMR (400 MHz, DMSO-d ₅ ) (subscript A = major rotamer, subscript B = minor rotamer; integrals are shown only for major rotamer) δ _Η 11.46 (s, 1H, NH _A ), 11.38 (s, NH _B ), 7.56 (s, 1H, H6 _A ), 7.52 (s, H6 _B ), 7.46 - 7.41 (m, 2H, Ar), 7.37 - 7.22 (m, 7H, Ar), 6.94 - 6.89 (m, 4H, Ar), 5.93 (d, J = 4.1 Hz, 1H, 3'-OH _A ), 5.89 (d, J = 4.5 Hz, 3'-OH _B ), 5.44 (s, 1H, Η1Ά), 5.39 (s, HI' _B ), 4.69 (s, H2' _B ), 4.41 (s, 1H, H2' _A ), 4.21 (m, 1H, H3'), 3.74 (s, 6H, 2 x OCH ₃ ), 3.47 - 3.28 (m, 4H, H5', H5"), 2.76 (t, J = 2.3 Hz, 1H, C≡CH), 2.72 - 2.52, 2.44 - 2.35 (m, 4H, 2 x CH ₂ ), 1.49 (s, 3H, 5-Me _A ), 1.46 (s, 1H, 5-Me _B ); ¹³ C NMR (101 MHz, DMSO- d ₆ ) 5c 169.4, 169.3, 163.9, 163.8, 158.2, 150.0, 149.8, 144.64, 144.58, 135.32, 135.25, 135.04, 135.01, 134.1, 129.8, 129.7, 128.0, 127.7, 126.9, 113.3, 108.6, 108.5, 87.9, 87.3, 86.6, 86.2, 85.8, 85.7, 83.9, 83.8, 71.3, 71.2, 69.2, 68.2, 63.0, 60.7, 59.3, 59.2, 55.1, 54.9, 51.1, 32.5, 32.0, 13.6, 13.4, 12.29, 12.25; HRMS-ESI m/z: 674.2454 ([M + Na] ⁺ ,

C3 ₇ H ₃ 7N ₃ 0 ₈ -Na ⁺ calcd 674.2473).

(lR,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)-phosphin oxy)-l-(4,4'- dimethoxytrityloxymethyl)-5-(pent-4-ynoyl)-3-(thymin-l-yl)-2 -oxa-5- azabicyclo[2.2.1]heptane (3). Nucleoside 2 (176 mg, 0.27 mmol) was co-evaporated with anhydrous CH ₂ CI ₂ , and subsequently mixed with Λ,/V-diisopropylammonium tetrazolide (69 mg, 0.40 mmol). The solids were dissolved in anhydrous CH ₂ CI ₂ (4.0 mL) and 2-cyanoethyl- Λ ,Λ ,Λ ',Λ '-tetraisopropylphosphane (120 μΙ_, 0.38 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 18 h, and then additional Λ ,/V- diisopropylammonium tetrazolide (49 mg, 0.29 mmol) and 2-cyanoethyl-/V,/V,/V',/V'- tetraisopropylphosphane (90 μΙ_, 0.28 mmol) were added and stirred for a further 5 h.

Ethanol (1 mL) was added and the resulting solution stirred for 20 min. The reaction mixture was then diluted with CH ₂ CI ₂ (20 mL) and washed with a saturated aqueous solution of NaHC0 ₃ (2 x 15 mL). The combined aqueous phases were back-extracted with CH ₂ CI ₂ (3 x 15 mL), and the combined organic phases were dried over Na ₂ S0 ₄ , filtered and the solvent removed in vacuo. The residue was purified by silica gel column chromatography (20-75% ethyl acetate/petroleum ether) giving a white foam. The foam was dissolved in ethyl acetate (1.5 mL) and the resulting solution was added dropwise to petroleum ether (150 mL). The formed precipitate was isolated affording phosphoramidite 3 as white foam (167 mg, 73%). R _f 0.63 (ethyl acetate); ³¹ P NMR (162 MHz, DMSO-d ₅ ) δ _Ρ 148.3, 147.9, 147.6, 147.0; HRMS- ESI m/z: 874.3528 ([M + Na] ⁺ , C ₄ 6H ₅ 4N ₅ 09P-Na ⁺ calcd 874.3551).

A. Fluorescent probes

Table SI. Photophysical characteristics of the single molecule dyes used in this study. ³

£max 3t λ ^6Χ ,

Dye λ ^6Χ , nm A ^f, _max , nm <D _F

cm^M ¹

5-R110 (M2) 496 520 80.000 0.9

Cy3 (M3) 546 563 150.000 0.1

Cy5 (M4) 646 662 250.000 0.2

28.000,

Perylene (M5) 408, 436 435, 463, 495 0.95

37.000

³ This information is available online, e.g. on web-page of fluorescent dyes supplier

Lumiprobe LLC: http://www.lumiprobe.com/

Post-synthetic click chemistry

Concentrations of oligonucleotides were calculated using the following extinction coefficients (OD ₂₅₀ / mol) : G, 10.5; A, 13.9; T, M ¹ , 7.9; C, 6.6; M ² , 11.8; M ³ , 5.7; M ⁴ , 4.3; 33.2; M ⁵ . General method for CuAAC reactions (monomers M ² -M ⁴ ). Starting oligonucleotide ON1-ON4 (20 nmol) was dissolved in fresh MQ water (30 μΙ_) in 1.5 mL plastic eppendorf. DMSO (40 μΙ_), 2 M triethylammonium acetate buffer (pH 7.4; 10 μΙ_), corresponding azide 3-6 (6 μΙ. (ONI), 10 μΙ. (ON2-ON3) and 14 μΙ_ (ON4) of 10 mM solution in DMSO), ascorbic acid (5 μΙ_ of 25 mM freshly prepared stock solution) and Cu(II)-TBTA equimolar complex (5 μΙ_ of 10 mM stock solution) were subsequently added. The resulting mixture was deaerated, tightly closed, mixed on vortex and left at rt (monomer M ² ; 12 h, ON1-ON3; 24 h, ON4). In case of highly hydrophobic cyanine azides 4-5 the reaction mixture was initially heated to 85 °C for 10 minutes and then left at rt for 12 h (ON1-ON3) or 24 h (ON4). The reaction was afterwards filtrated through Illustra NAP-10 column (GE Healthcare) following manufacture's protocol. The resulting solution was evaporated followed by precipitation of the product conjugates from cold acetone (-18 °C, 12 h) and subsequent washing with acetone two times. The resulting conjugates ON5-ON16 were analyzed by MALDI TOF mass spectrometry and IE HPLC (Table S2). Final yields of products based on the absorbance at 260 nm : 85% (ON5), 78% (ON6), 80% (ON7), 74% (ON8), 63% (ON9), 58% (ONIO), 63% (ON11), 62% (ON12), 57% (ON13), 58% (ON14), 60% (ON15), 54% (ON16) .

General method for microwave-assisted CuAAC reactions (monomer M ⁵ ). Starting oligonucleotide ON1-ON4 (20 nmol) was dissolved in fresh MQ water (30 μΙ_) in 1.5 mL plastic eppendorf. DMSO (40 μΙ_), 2 M triethylammonium acetate buffer (pH 7.4; 10 μΙ_), corresponding azide 3-6 (6 μΐ (ONI), 10 μΐ (ON2-ON3) and 14 _μ Ι_ (ON4) of 10 mM solution in DMSO), ascorbic acid (5 μΙ_ of 25 mM freshly prepared stock solution) and Cu(II)- TBTA equimolar complex (5 μΙ_ of 10 mM stock solution) were subsequently added. The resulting mixture was deaerated, tightly closed, mixed on vortex and subjected to microwave conditions (microwave reactor, 60 °C, 15 minutes). The reaction was afterwards cooled to room temperature and filtrated through Illustra NAP-10 column (GE Healthcare) following manufacture's protocol. The resulting solution was evaporated followed by precipitation of the product conjugates from cold acetone (-18 °C, 12 h) and subsequent washing with acetone two times. The resulting conjugates ON17-ON20 were analyzed by MALDI TOF mass spectrometry and IE HPLC (Table S2). Final yields of products based on the absorbance at 260 nm : 81% (ON17), 72% (ON18), 76% (ON19), 65% (ON20).

Table S2. IE HPLC retention times and MALDI MS of purified oligonucleotides. ³

# Sequence, 5'→3' Ret. time, MALDI MS

mm

Found m/z Calc. m/z

[M-H] ^" [M-H] ^" ON I TGC ACT CTA TGM ¹ CTG TAT CAT 8^88 6467 6468

ON2 TGC ACT CTA M ^X GT CM^ TAT CAT 8.91 6575 6575

ON3 TGC ACM ¹ CTA TGT CTG TAM ¹ CAT 8.90 6572 6575

ON4 TGC ACM ¹ CTA TGM ¹ CTG TAM ¹ CAT 8.91 6680 6682

ON5 TGC ACT CTA TGM ² CTG TAT CAT 9Λ0 6925 6925

ON 6 TGC ACT CTA M ² GT CM ² G TAT CAT 9.13 7491 7488

ON7 TGC ACM ² CTA TGT CTG TAM ² CAT 9.13 7489 7488

ON8 TGC ACM ² CTA TGM ² CTG TAM ² CAT 9.34 8053 8052

ON9 TGC ACT CTA TGM ³ CTG TAT CAT 10.00 7008 7008

ON IO TGC ACT CTA M GT CM G TAT CAT 10.01 7657 7655

ON 11 TGC ACM ³ CTA TGT CTG TAM ³ CAT 10.20 7655 7655

ON 12 TGC ACM ³ CTA TGM ³ CTG TAM ³ CAT 10.42 8300 8302

ON 13 TGC ACT CTA TGM ⁴ CTG TAT CAT 11.40 7031 7034

ON 14 TGC ACT CTA M ⁴ GT CM ⁴ G TAT CAT 11.45 7707 7707

ON 15 TGC ACM ⁴ CTA TGT CTG TAM ⁴ CAT 11.50 7706 7707

ON 16 TGC ACM ⁴ CTA TGM ⁴ CTG TAM ⁴ CAT 11.93 8381 8379

ON 17 TGC ACT CTA TGM ⁵ CTG TAT CAT 9Λ Ϊ 6800 6803

ON 18 TGC ACT CTA M ⁵ GT CM ⁵ G TAT CAT 9.11 7242 7245

ON 19 TGC ACM ⁵ CTA TGT CTG TAM ⁵ CAT 9.20 7244 7245

ON20 TGC ACM ⁵ CTA TGM ⁵ CTG TAM ⁵ CAT 9.39 7688 7687 Steady-state fluorescence emission spectra of ON5-ON20 and their duplexes with complementary DNA/RNA were recorded in a medium salt buffer at 19 °C using characteristic excitation wavelengths of the incorporated dyes (Figure 1) .

Autoantibody binding assay

General

The human monoclonal autoantibodies were purchased from Diarect AG (dsDNA-mAb32 and dsDNA-mAb33 correspond to clones 32. B9 and 33.H11, respectively). BSA was obtained from Sigma-Aldrich and dissolved in a medium salt PBS at concentration 1.5 mg/mL.

Incubation and Analysis

To a solution of corresponding nucleic acid complex prepared as described above in a plastic 1.5 mL tube (500 μΙ_ 0.5 μΜ), 0.5χ10 ⁵ IU of the target autoantibody was added (53 μΙ_, dsDNA-mAb32 9.4x l0 ⁵ IU/mL at a protein concentration 0.43 mg/mL; 128 μί, dsDNA-mAb33 3.9x l0 ⁵ IU/mL at a protein concentration 0.70 mg/mL). As a reference, 33 μί of the BSA stock solution was used in similar incubation reactions with the probes. Incubation was performed on Eppendorf Thermomixer Shaker (400 rpm) at 37 °C for 3 h. Upon cooling to ambident temperature over 1 h, the resulting solutions were analyzed by fluorescence spectroscopy using λ ^6Χ 500 nm and monitoring A ^l 530 nm.

Limit of target detection (LOD) values were determined by series of incubations and subsequent analysis of dsDNA-mAb33 at concentrations (χΐθ ⁵ ) : 1 IU/mL, 0.5 IU/mL, 0.25 IU/mL, 0.1 IU/mL, 0.05 IU/mL, 0.01 IU/mL, and ON7: DNA at concentration 0.5 μΜ as described above. Resulting fluorescence intensities revealed LOD < 2.5χ10 ³ IU/mL, corresponding to the autoantibody concentration < 4.6 μg/mL.

Table S3. Fluorescence detection of antibody binding by single-stranded

oligonucleotides and duplexes containing monomer M ² .

Homogeneous detection of human autoantibodies

An important advantage of synthetic oligonucleotide within molecular diagnostics of proteins (so-called aptasensing approach) and appealing immunoimaging techniques is their high specificity and possibility of the assay's standardization, which is hard to achieve using highly heterogeneous natural sensors. To assess the potential of the novel probes in diagnostics of clinically important protein targets, fluorescence homogeneous detection of human autoantibodies against double-stranded DNA was performed. Single-stranded ON7-ON8 and their duplexes with complementary DNA/RNA were incubated with commercially available human monoclonal autoantibodies dsDNA-mAb32 and dsDNA-mAb33, which were recently studied by surface plasmon resonance (SPR).

Incubation was performed in a medium salt phosphate buffer (pH 7.0) at 37 °C for 3 h followed by fluorescence analysis after 1 h at 19 °C. In order to evaluate the probes' specificity BSA protein was used as a reference target. The double-stranded complex

ON7: DNA showed binding to exclusively dsDNA-mAb33 confirmed by 1.7-fold increase of fluorescence at 530 nm and 240% superiority of the fluorescence with respect to dsDNA- mAb32 target (Figure 2). Previously SPR studies indicated fast dissociation rate for the autoantibody dsDNA-mAb33 compared to dsDNA-mAb32 in binding 24 bp DNA duplex ((k _d ) ₀ bs ~ 6.5xl0 ^"3 s ^"1 and 0.5χ10 ^"3 s ^"1 , respectively). Fluorescence binding pattern by ON7: DNA implies that chemical modification of the double-stranded DNA remarkably changes binding properties to the specific protein targets. Notably, no fluorescence signal of interaction with BSA was observed for ON7: DNA confirming no cross- reactivity for the prepared nucleic acid complex compared to single-stranded ON7, ON7: RNA and to triply modified ON8: DNA/RNA (Table S3, above). Effective recognition of dsDNA-mAb33 is provided by steric and chemical complementarity of the unmodified internal region of ON7: DNA and heavy chain of the autoantibody, accompanied by effective hydrogen bonding.

Finally, limit of target detection (LOD) value for ON7: DNA was below 4.6 pg/mL of dsDNA- mAb33. This is comparable with currently applied enzyme-linked immunosorbent assay (ELISA), immunofluorescence tests (LOD approx. 1-2 pg/mL), and other fluorescent aptasensors. Importantly, being compared to voltage current and electrochemical signal methods, homogeneous detection is rapid and furthermore does not affect interacting surfaces of the biomolecules, which can be detected in a direct way, without the need for additional steps and reagents.

B. Peptide-Oligonucleotide Conjugates (POCs)

Post-synthetic click chemistry

Concentrations of oligonucleotides were calculated using the following extinction coefficients OD260/pmol) : G, 10.5; A, 13.9; T, Ml, 7.9; C, 6.6; M6, 8.1 ; M7, 9.2. Extinction coefficients at 260 nm of monomers M6-M7 were determined by summarizing extinction coefficient of monomer Ml and the corresponding azide 6-7. The latter values were measured at 19 °C in 5% DMSO-water, v/v.

General method for CuAAC reactions. Starting oligonucleotide ON1-ON3 (20 nmol) was dissolved in fresh MQ water (30 μΙ_) in a 1.5 mL plastic eppendorf. DMSO (40 μΙ_), 0.2M carbonate buffer (pH 8.5; 10 μΙ_), corresponding azide 6-7 (6 μΙ_ (ONI) and 10 μΙ_

(ON2-ON3) of 10 mM solution in 30 % DMSO-0.02M carbonate buffer), aminoguanidine hydrochloride (5 μΙ_ of 50 mM freshly prepared stock solution), 6 ascorbic acid (5 μΙ_ of 25 mM freshly prepared stock solution) and Cu(II)-TBTA equimolar complex (5 μΙ_ of 10 mM stock solution) were subsequently added. The resulting mixture was deaerated, tightly closed, mixed on vortex and left at rt for 12 h (ONI), or 24 h (ON2-ON3) . The reaction was afterwards filtrated through Illustra NAP-10 column (GE Healthcare) following manufacture's protocol. The resulting solution was evaporated followed by precipitation of the product conjugates from cold acetone (-18 °C, 12 h) and subsequent washing with acetone two times. The resulting conjugates POC1-POC24 (= SEQ ID No. 21-44) were analyzed by MALDI-TOF mass spectrometry and IE HPLC (see Table S2). Final yields of the products based on absorbance at 260 nm: 88% (POC1), 84% (POC2), 78% (POC3), 70% (POC4), 82% (POC5), 79% (POC6).

Table S2. IE HPLC retention times and MALDI-MS of POC1-POC6 # Sequence, 5'→3' Ret. time, MALDI MS

min

Found m/z Calc. m/z

[M-H] ^" [M-H] ^"

POC1 TGC ACT CTA TGM ⁶ CTG TAT CAT 22.91 7550 7551

POC2 TGC ACT CTA M ⁶ GT C M ⁶ G TAT CAT 21.13 8748 8741

POC3 TGC ACM ⁶ CTA TGT CTG TAM ⁶ CAT 21.19 8745 8741

POC4 TGC ACT CTA TGM ⁷ CTG TAT CAT 23.54 7533 7533

POC5 TGC ACT CTA M ⁷ GT CM ⁷ G TAT CAT 21.25 8708 8705

POC6 TGC ACM ⁷ CTA TGT CTG TAM ⁷ CAT 23.24 8706 8705

POC7 TGC ACT CTA TGM ⁸ CTG TAT CAT 22.30 7480 7483

POC8 TGC ACT CTA M ⁸ GT CM ⁸ G TAT CAT 21.10 8606 8606

POC9 TGC ACM ⁸ CTA TGT CTG TAM ⁸ CAT 21.13 8604 8606

POCIO TGC ACT CTA TGM ⁹ CTG TAT CAT 21.15 7465 7465

POC11 TGC ACT CTA M ⁹ GT CM ⁹ G TAT CAT 20.32 8577 8570

POC12 TGC ACM ⁹ CTA TGT CTG TAM ⁹ CAT 20.30 8578 8570

POC13 TGC ACT CTA TGM ¹⁰ CTG TAT CAT 19.99 7094 7099

POC14 TGC ACT CTA M ¹⁰ GT CM ¹⁰ G TAT 18.92 7837 7838

CAT

POC15 TGC ACM ¹⁰ CTA TGT CTG TAM ¹⁰ 18.19 7838 7838

CAT

POC16 TGC ACT CTA TGM ¹¹ CTG TAT CAT 19.04 7080 7081 POC17 TGC ACT CTA M"GT CM"G TAT 18.23 7800 7802

CAT

POC18 TGC ACM ¹¹ CTA TGT CTG TAM ¹¹ 18.02 7799 7802

CAT

POC19 TGC ACT CTA TGM ¹² CTG TAT CAT 27.90 7866 7865

POC20 TGC ACT CTA M ¹² GT CM ¹² G TAT 29.91 9373 9370

CAT

POC21 TGC ACM ¹² CTA TGT CTG TAM ¹² CAT 28.22 9371 9370

POC22 TGC ACT CTA TGM ¹³ CTG TAT CAT 31.11 8640 8644

POC23 TGC ACT CTA M ¹ GT CM ¹ G TAT 33.23 10924 10928

CAT

POC24 TGC ACM ¹³ CTA TGT CTG TAM ¹³ 33.15 10925 10928

CAT

M ⁶ and M ⁷ are long-chain enkefalins with K residues attached to LNA monomers, namely 5'- azidopentanoic acid-KKKYGGFM (M ⁶ ) and 5'-azidopentanoic acid-KKKYGGFL (M ⁷ ) .

Monomer M : 5'-azidopentanoic acid-KKKYGGFM-CONH ₂

Monomer M ⁹ : 5'-azidopentanoic acid-KKKYGGFL-CONH ₂

Monomer M ¹⁰ : 5'-azidopentanoic acid-YGGFM-CONH ₂

Monomer M ¹¹ : 5'-azidopentanoic acid-YGGFL-CONH ₂

Monomer M ¹² : 5'-azidopentanoic acid-RKKRRQRRR-CONH ₂

Monomer M : 5'-azidopentanoic acid-RQIKIWFQNRRMKWK-CONH ₂

DNA reference info (figures 3A and 3B) : 5'-d(TGC ACT CTA TGT CTG TAT CAT),

MS calc. 6361 (M-H-); MS found: 6361.

Ret. Time (IEHPLC) 18.22

Serum stability assay

To a solution of starting oligonucleotide (2.9 μΙ_ of 10 μΜ stock solution) in a per tube the corresponding 32P-labeled analogue (2.0 μΙ_ of 0.5 μΜ solution) in mQ water and human serum (HS: 16 μΙ_; 90% or 10% in HBSS buffer, pH = 7.4) were added . Pre-treatment of the human serum with 1 mM solution of paraoxon-ethyl was performed as described in literature (O. S. Gudmundsson, K. Nimkar, S. Gangwar, T. Siahaan, R. T. Borchardt, Pharm. Res., 1999, 16, 16) . Incubation was performed on Eppendorf Thermomixer Shaker (300 rpm) at 37 °C. The alicvotes were taken in 2 min, 5 min, 10 min, 30 min, and in 1 h, 4 h, 8 h and 24 h time intervals after beginning of the assay, quenching each alicvote with a 1 vol ice-cold 95% formamide with excess EDTA. The resulting probes were analyzed on 13 % denaturing polyacrilamide gels (7 M urea; IX TBE) . Visualization was performed by autoradiography on Typhoon Trio Variable Mode Imafer (Amersham Biosciences) . Figures 3A-3D show gel electrophoresis of 5'- ³² P-labelled oligonucleotides incubated with HS.

The peptide-modified analogues POC2 (SEQ ID No. 22) and POC5 (SEQ ID NO. 25) demonstrated a significant resistance to enzymatic attack in 90% HS (> 8 h; Figure 3A-D) .

ASPECTS OF THE INVENTION

The invention relates to the following aspects:

Aspect 1 : An oligonucleotide comprising an alkyne-LNA nucleotide unit of formula (I) :

(I) wherein B is a purine or pyrimidine nucleobase; and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

Aspect 2: The oligonucleotide according to aspect 1, wherein A is -(C=0)-(CH ₂ ) _n -, in which n = 1-5.

Aspect 3 : The oligonucleotide according to any one of the preceding aspects, wherein B is a pyrimidine nucleobase.

Aspect 4: The oligonucleotide according to any one of the preceding aspects, comprising a sequence with SEQ ID No. 1-4, preferably SEQ ID No. 2, 3 or 4.

Aspect 5 : A method for synthesizing a fluorescent LNA oligonucleotide, said method comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a fluorescent dye compound having the structure:

FL-N ₃ in which FL constitutes a fluorescent moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the fluorescent dye compound having the structure FL-N ₃ so as to form a 1,2,3-triazole product.

Aspect 6: A fluorescent LNA oligonucleotide, comprising a fluorescent-LNA nucleotide monomer of formula (II) :

(Π) wherein B is a purine or pyrimidine nucleobase; FL constitutes a fluorescent moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

Aspect 7: The fluorescent LNA oligonucleotide according to aspect 6, wherein A is -(C=0)- (CH ₂ ) _n -, in which n = 1-5.

Aspect 8: The fluorescent LNA oligonucleotide according to any one of aspects 6-7, wherein B is a pyrimidine nucleobase.

Aspect 9: The fluorescent LNA oligonucleotide according to any one of aspects 6-8, wherein FL comprises a cyanine, perylene, pyrene or other PAH, or a xanthene fluorescent moiety.

Aspect 10: The fluorescent LNA oligonucleotide according to any one of aspects 6-9, comprising a sequence with SEQ ID No. 5-20, preferably SEQ ID No. 7, 8, 20, most preferably SEQ ID No. 7.

Aspect 11 : A duplex formed between the fluorescent LNA oligonucleotide according to any one of aspects 6-10, and the complementary DNA/RNA.

Aspect 12: Use of the fluorescent LNA oligonucleotide according to any one of aspects 6-10 as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA.

Aspect 13 : Use of the fluorescent LNA oligonucleotide according to any one of aspects 6-10, or the duplex according to aspect 11, for the detection of an autoimmune antibody.

Aspect 14: A method for synthesizing a peptide-oligonucleotide conjugate (POC), said method comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a peptide compound having the structure:

Q-N ₃ in which Q constitutes a peptide moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the peptide compound having the structure Q-N ₃ so as to form a 1,2,3-triazole product. Aspect 15 : A peptide-oligonucleotide conjugate (POC) comprising a peptide-LNA

oligonucleotide monomer of formula (III) :

(III) wherein B is a purine or pyrimidine nucleobase; Q constitutes a peptide moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

Aspect 16: The peptide-oligonucleotide conjugate (POC) according to aspect 15, wherein A is -(C=0)-(CH ₂ ) _n -, in which n = 1-5.

Aspect 17: The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-

16, wherein B is a pyrimidine nucleobase.

Aspect 18: The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-

17, wherein Q is a Met or Leu- enkephalin derivative containing additional lysine residues at the N-terminus.

Aspect 19: A duplex formed between the peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-18, and the complementary DNA/RNA.

Aspect 20: Use of the peptide-oligonucleotide conjugate (POC) according to any one of aspects 15-18 as a LNA/DNA probe for the detection of complementary DNA and/or RNA.

Aspect 21 : The peptide-oligonucleotide conjugate (POC) according to any one of aspects 15- 18 for use in therapy, in particular antisense therapy.

Aspect 22: A method for synthesizing a carbohydrate-oligonucleotide conjugate (COC), said method comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a carbohydrate compound having the structure: in which CHO constitutes a carbohydrate moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the carbohydrate compound having the structure CHO- N3 so as to form a 1,2,3-triazole product.

Aspect 23 : A carbohydrate-oligonucleotide conjugate (COC) comprising a carbohydrate-LNA oligonucleotide monomer of formula (IV) :

(IV)

wherein B is a purine or pyrimidine nucleobase; Q constitutes a peptide moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted C ₁ -C ₁ 0 alkyl, or combinations thereof.

Aspect 24: A method for synthesizing a lipid-oligonucleotide conjugate (LOC), said method comprising the step of reacting the oligonucleotide according to any one of aspects 1-4 with a lipid compound having the structure: in which L constitutes a lipid moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne- LNA nucleotide of formula (I) reacts with the azide moiety of the lipid compound having the structure L-N ₃ so as to form a 1,2,3-triazole product.

Aspect 25 : A lipid-oligonucleotide conjugate (LOC) comprising a lipid-LNA oligonucleotide monomer of formula (V) :

wherein B is a purine or pyrimidine nucleobase; Q constitutes a peptide moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

Aspect 26: A method for synthesizing a protein-oligonucleotide conjugate, said method comprising the step of reacting the oligonucleotide according to any one of aspect 1-4 with a protein compound having the structure:

Pro-N ₃ in which Pro constitutes a protein moiety and N ₃ constitutes an azide moiety, wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition in which the alkyne moiety of the alkyne-LNA nucleotide of formula (I) reacts with the azide moiety of the protein compound having the structure Pro-N ₃ so as to form a 1,2,3-triazole product.

Aspect 27: A protein-oligonucleotide conjugate comprising a protein-LNA oligonucleotide monomer of formula (VI) :

(VI)

wherein B is a purine or pyrimidine nucleobase; Q constitutes a peptide moiety and A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally-substituted Ci-Ci ₀ alkyl, or combinations thereof.

Aspect 28: An alkyne-LNA nucleoside of formula (X) : wherein B is a purine or pyrimidine nucleobase; PI is H or a protecting group; P2 is H, a protecting group or a coupling group, and A is selected from a single bond, -(C=0)-, -0-, - NH-, -S- and optionally-substituted Ci-Cio alkyl, or combinations thereof.

Aspect 29: An alkyne-LNA nucleoside of formula (X) according to aspect 28, wherein PI is protecting group, e.g. DMT, MMT or Trityl (Tr).

Aspect 30: An alkyne-LNA nucleoside of formula (X) according to aspect 28-29, wherein P2 is coupling group, e.g. a phosphoramidite, e.g. -P(N(Pr) ₂ )OC ₂ H ₄ CN, a phosphodiester or phosphotriester.