FIELD OF THE INVENTION

The present invention relates to novel approach to the design and preparation of effective fluorescent probes for RNA biosensing. Rationally designed 2'-(H-l,2,3-triazoyl)-methoxy oligonucleotides are provided for fluorescence-based bioanalysis. These can be readily synthesised via "click chemistry" on the corresponding oligonucleotides comprising 2'-0- alkylnyl nucleotides.

BACKGROUND OF THE INVENTION Herein a novel approach to the design and preparation of effective fluorescent probes for DNA/RNA biosensing is described. The probes have been prepared by postsynthetic CuAAC click chemistry using series of commercially-available fluorescent azides and alkyne-modified oligonucleotide precursors. Fluorescent signalling of hybridization by internally positioned polyaromatic hydrocarbons and xanthene dyes was achieved with low fluorescence background signal, remarkably high fluorescence quantum yields at ambient and elevated temperature, accompanied by high selectivity and signal specificity of the probes when binding to natural RNA targets.

RNA targeting is a rapidly evolving field of research and clinical diagnostics which applies synthetic fluorescent probes and modern biophysical techniques to monitor desired targets in vitro and in vivo. Preparation of fluorescent oligonucleotides with high potential in biologically relevant DNA/RNA biosensing by a simple and rapid method, e.g. by fluorescence, is an important aspect of current nucleic acid chemistry. Recently copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has been successfully applied for nucleic acid labelling, allowing preparation of various oligonucleotides in high yields and of remarkable purity. However, in order to further utilize "clickable" oligonucleotides in diverse biosensing experiments, the fluorescent probes have to meet several additional requirements, such as high photo- and chemical stability, low fluorescence background signal of single strands, high binding specificity and low limit of target detection. Ideally, incorporation of fluorescent dyes should be simple and allow high flexibility in probe design. WO2010/129656 discloses a method for oligonucleotide labelling using click chemistry, in which an oligonucleotide having a 2' O-alkynyl substituent is reacted with a "reporter group" having an -N ₃ moiety. The aim of this publication is to provide FRET pairs.

Berndl et al. Bioconjugate Chem., 2009, 20, 558-564 discloses click chemistry between propyne-derived oligonucleotides and N ₃ -functionalised fluorescent moieties.

WO2013/013068 discloses monomers which are incorporated into DNA sequences. The monomers arise from click chemistry. Probes, and duplexes thereof, including two DNA moieties are also disclosed.

K. Astakhova & al, Chem., Eur . 2013, 19, 1112-1122 discloses click chemistry between a DNA sequence having clickable deoxyribose units and N ₃ -functionalised fluorescent moieties. Solid phase chemistry is used to obtain two different fluorescent moieties on the same DNA sequence, which can thereby function as FRET pairs.

MicroRNAs (miRNAs) are short (~22 nucleotides) noncoding RNA molecules that post- transcriptionally repress the expression of protein-coding genes by binding to 3'-untranslated regions of the target mRNAs. An increasing amount of evidence shows that miRNAs control a large number of biological processes, including cell differentation, tumourigenesis and the regulation of metabolism. Furthermore, miRNA-7 (miR-7) were recently identified to be selectively expressed within the hypothalamus, a part of the brain that controls vital bodily functions. Another recently discovered RNA molecules with important, yet not fully studied, regulatory functions are circular RNAs (circRNAs). Interestingly, it was found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDRlas), is densely bound by microRNA (miRNA) effector complexes and multiple conserved binding sites for the ancient miRNA miR-7. Further analyses of miR-7 and circRNAs will significantly contribute current understanding of regulatory RNA pathways and possibly help discovery of previously unrecognized regulatory potential of coding sequences.

SUMMARY OF THE INVENTION

So, in a first aspect the present invention relates to a fluorescent oligonucleotide, comprising two or more fluorescent 2'-0-substituted nucleotide monomers of formula (II) located internally in said oligonucleotide:

(Π) wherein B is a purine or pyrimidine nucleobase, A is a linker moiety and wherein each FL constitutes the same fluorescent moiety. Duplexes formed between such a fluorescent oligonucleotide and the complementary DNA or RNA are also provided. The use of such a fluorescent oligonucleotide as a fluorescent LNA/DNA probe for the detection of

complementary DNA and/or RNA, or for the detection of an autoimmune antibody, is provided.

Furthermore, an oligonucleotide is provided comprising one or more 2'-0-alkylnyl nucleotide units of formula (I) :

(I)

wherein B is a purine or pyrimidine nucleobase and A is a linker moiety. A method for synthesizing a fluorescent oligonucleotide is provided, according to claim 26, and a method for designing and synthesising a fluorescent oligonucleotide probe is provided, according to claim 27.

Further details of the invention are provided in the dependent claims, and in the following detailed disclosure of the invention. LEGENDS TO THE FIGURES

Fig. 1 General strategy for design of probes targeting natural RNA targets (i .e. miR-7 and circRNA) ; chemical structure of 2'-0-propargyl uridine monomer U ^p .

Fig. 2 Representative visible absorbance spectra of modified oligonucleotides (not normalized) . Spectra were obtained in medium salt buffer at 19 °C using 1.0 μΜ

concentrations of single-stranded oligonucleotides.

Fig. 3. Thermal denaturation curves of duplexes containing monomers M*-M ³ and unmodified references recorded in a medium salt phosphate buffers using 1.0 μ Μ

concentration of oligonucleotides. Fig 4. Representative CD spectra of reference oligonucleotides (ON4 and R6) and fluorescent probes P2 and P4 against complementary circRNA. The spectra were recorded in medium salt buffer at 19 °C, using 2.0 μΜ concentration of complementary strands.

Reference strand R6 : 5 '- ( 6 F A M ) - d ( G A AG AC ^L GTG ^L G ATTTTCTG G A ^L AG A) , obtained from Exiqon.

Fig. 5. Thermal denaturation temperatures at 260 nm for references (A) and "clickable" probes (B) binding circRNA targets; AT _m is the difference between the T _m values of a fully complementary and a mismatched complex. The first bar in each series corresponds to fully complementary probe: RNA complex with AT _m = 0 (shown as a grid bar) . Reference probes R1-R4 and mismatched RNA targets (MT1-MT8 left to right) are listed in Table S2.

Fig. 6. Representative steady-state fluorescence emission spectra of single-stranded probes (black lines), and their duplexes with complementary DNA and RNA (shown in dotted and doubled lines, respectively) . Spectra were obtained in a medium salt buffer at 19 °C using excitation wavelength of 425 nm (A,B) and 480 nm (C,D) .

Fig. 7. Representative fluorescence sensing of complementary and mismatched circRNA targets by the references and "clickable" probes prepared in this study. The first two bars in each series correspond to single stranded probe and its complex with fully complementary RNA) . Reference probes R1-R4 and mismatched RNA targets (MT1-MT8 left to right) are listed in Table S2. Spectra were obtained in a medium salt buffer at 19 °C using 1.0 μΜ concentrations of the complementary strands and the following excitation and emission wavelengths: 425/452 nm (P2) 480/530 nm (P4), 580/605 nm (P6), 494/520 nm (R5, R6), 533/550 nm (R7), and 494/519 nm (R8) . DETAILED DISCLOSURE OF THE INVENTION

In the present work a series of fluorescent probes containing internally positioned

ultrasensitive polyaromatic hydrocarbons (PAHs) and xanthene dyes was prepared in order to achieve high binding affinity and efficient fluorescent sensing of miR-7 and circRNA in vitro (Fig. 1). Herein, the synthesis of miR-7 and circRNA targeting oligonucleotides containing 2'- O-propargyl uridine scaffolds U ^p , their fluorescent labelling by CuAAC click reactions, and comprehensive spectroscopic characterization of the resulting fluorescent probes in vitro are reported. Attachment of fluorophores by click chemistry to 2'-0-propargyl uridine scaffold U ^p was shown to give probes with promising properties in biosensing (Fig. 1).

In a first aspect, therefore, the invention provides a fluorescent oligonucleotide, comprising two or more fluorescent 2'-0-substituted nucleotide monomers of formula (II) located internally in said oligonucleotide:

(Π) wherein B is a purine or pyrimidine nucleobase, preferably a pyrimidine nucleobase, e.g. uracil (U), A is a linker moiety and wherein each FL constitutes the same fluorescent moiety.

The fluorescent oligonucleotide may comprise two or three 2'-0-substituted nucleotide monomers of formula (II). Fluorescent oligonucleotides with more fluorescent moieties, showed poorer light up of fluorescene when binding to the targets, lower quantum yields and worse binding affinity and selectivity to targets.

The phrase "located internally in said oligonucleotide" is used in the context of the present invention to mean that the 2'-0-substituted nucleotide monomers of formula (II) are not located at the 5' or the 3' ends of the oligonucleotide.

WO2010/129656 discloses oligonucleotides which include fluorescent moieties located only at the 5'-terminal end of the oligonucleotide (cf. Table 2). This is a consequence of the method by which said oligonucleotides are synthesised. However, terminally-labelled FRET pairs as shown are not sensitive to SNP and therefore have no utility (see e.g. Jorgensen et al, Chemm Commun 2013).

WO2010/129656 describes application of the product probes when the quencher of fluorescence and the dye are attached. WO2010/129656 further describes different aspects of using quencher molecules for detecting nucleic acid hybridization. However

WO2010/129656 does not provide any details on probe design which could lead to the effective target detection, except for utilization of a dye-quencher principle. Quenchers are often chemically and photo-unstable, expensive organic molecules. Moreover, additional sequences have to be incorporated in order to apply the dye-quencher mechanism. This includes necessity to attach the stem for molecular beacons, or avoid certain bases opposite to the fluorophores. Suitably, therefore, the oligonucleotides of the present invention do not comprise quencher moiety or moieties.

As confirmed by our studies, a person skilled in the state-of-the-art cannot use the

WO2010/129656 to prepare quencher-free probes by click chemistry, which will effectively sense complementary target (DNA/RNA or both). Moreover, single-nucleotide polymorphism (SNP) sensing is not mentioned in WO2010/129656. SNP detection and analysis is one of the major diagnostic and research fields in nucleic acid targeting.

The fluorescent oligonucleotide according to the invention may have a total length of between 10 and 30 nucleotides, preferably between 13 and 23 nucleotides.

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).

In structures I-II, A is a linker moiety; i.e. a moiety having dual connectivity which links the 2'-0 position of the ribose with the triazole ring. Suitably, A comprises between zero and 30 atoms. Suitably, A is selected from a single bond, -(C=0)-, -0-, -NH-, -S- and optionally- substituted Ci-Cio alkyldiyl, or combinations thereof. A may suitably consists of a

combination of 2-5 of the above-mentioned groups.

Suitably, A comprises -(C=0)-, and may form an ester with the O-atom of the ribose. A may be -(C=0)-(CH ₂ ) _n - G-e. it forms a straight-chain alkyl ester linker with the O-atom of the ribose) in which n = 1-5, such as 1 or 2, preferably 2. In the present context, the term "Ci_Ci ₀ alkyldiyl" is intended to mean a linear, cyclic or branched hydrocarbon group having 1 to 10 carbon atoms and two substituents, respectively, such as methydiyl, ethyldiyl, propyldiyl, /so-propyldiyl, cyclopropyldiyl, butyldiyl, iso- butyldiyl, tert-butyldiyl, cyclobutyldiyl, pentyldiyl, cyclopentyldiyl, hexyldiyl, and

cyclohexyldiyl. Preferred examples of Ci-Cio alkyldiyl as a part of linker A are Ci-C ₆ -alkyldiyl, such as Ci-C ₄ -alkyldiyl and Ci-C ₂ -alkyldiyl, such as Ci or C ₂ alkyldiyl . Preferred A is -CH ₂ -.

In the present context, i. e. in connection with the term "Ci-Ci ₀ -alkyldiyl", 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-6-alkyl-oxy), amino, mono- and di(Ci- ₆ -alkyl)amino, -N(Ci- ₄ -alkyl) ₃ ⁺ , cyano, nitro, Ci _-6 - alkylthio, and halogen. The term "halogen" includes fluoro, chloro, bromo, and iodo.

In some important embodiments, Ci-Ci ₀ -alkyldiyl (and any variants hereof) as a part of the group A is unsubstituted.

The fluorescent oligonucleotide of the invention may comprise two or more FL moieties, each being the same cyanine, perylene, pyrene or other PAH, or xanthene fluorescent moiety.

Other dyes and nanoparticles composed of the dyes or inorganic materials can be applied as well . Perylene and two xanthene derivatives, 5-R110 and 6-ROX, were selected as fluorophores with promising optical properties to be internally attached to the scaffold U ^p within new RNA targeting probes (Fig . 1 ; Scheme 1) . In doing this, commercially available phosphoramidite reagent 1 was triply incorporated into 23mer miR-7 and circRNA targeting sequences resulting in oligonucleotides ON 1-ON2 (Fig . 1, Table 1 ; Table SI)

Scheme 1. Modified monomers applied in this invention; postsynthetic CuAAC click chemistry generating fluorescent miR-7 and circRNA oligonucleotides.

The "clickable" scaffolds U ^p within ON1-ON2 were separated by 1-2 nucleotides in order to achieve optimal fluorescence light-up of the probes upon target binding . In one aspect, therefore, in the fluorescent oligonucleotide according to the invention, adjacent 2'-0- substituted nucleotide monomers of formula (II) are separated by 1 or 2 nucleotides A, C or T (not G) .

Additional LNA monomers were incorporated into ON 1-ON2 to enhance binding affinity and selectivity of the probes to complementary targets. Furthermore, ON3-ON4 were prepared as reference LNA/DNA probes in order to evaluate influence of the modified monomers U ^p and M*- M ³ on target binding (Table 1) . It has been discovered that control probes without LNA show worse performance than LNA/DNA mixmer probes. Suitably, therefore, the fluorescent oligonucleotides may comprise one or more locked nucleic acid (LNA) units, such as two or more LNA units, or three or more LNA units. Suitably, the two or more locked nucleic acid (LNA) units are separated by at least one other nucleotide A, C, T or G.

Preferably, the one or more LNA units are not located between two 2'-0-substituted nucleotide moieties of formula (II) in said oligonucleotide. Table 1 Thermal denaturation temperatures (T _m ) of the modified duplexes in a medium salt phosphate buffer. ³

VS.

# Sequence 5'→3' Target complement

ary

DNA RNA

ON I ACAACAAA^TC^CU^GU^U^CC^ miR-7 62.0 66.5

ON2 GAAGAC 3TG 3AU^U^CU ^P GGA ^L AGA circRNA 62.0 66.0

ON3 ACAACAAA ^L ATC ^L ACTAGTCTTCC ^L A miR-7 67.0 66.0

ON4 GAAGAC ^L I ^L GA I 1 1 1 1 GA ^L AGA circRNA 70.0 72.0

PI ACAACAAA'-ATC^CM^GM^M^CC^ miR-7 68.0 68.0

P2 GAAGAC ^L GTG ^L GA i ¹ TM ¹ TCM ¹ GGA ^L AGA circRNA 68.0 64.0

P3 ACAACAAA ^L ATC ^L ACM ² AGM ² CM ² TCC ^L A miR-7 57.0 62.0

P4 GAAGAC ^L GTG ^L GAM ² TM ² TCM ² GGA ^L AGA circRNA 57.0 61.0

P5 ACAACAAA ^L ATC ^L ACM AGM CM TCC ^L A miR-7 55.0 60.0

P6 GAAGAC ^L GTG ^L GAM TM TCM GGA ^L AGA circRNA 57.0 59.0

³ T _m of unmodified duplexes formed by miR-7 and circRNA: 61.0 °C and 60.0 °C with complementary DNA, and 57 °C and 55.5 °C with complementary RNA, respectively; LNA monomers are marked with an L in superscript.

In particular, the invention provides fluorescent oligonucleotides P1-P6 according to the invention. The fluorescent oligonucleotides (probes) prepared herein are demonstrated to be new promising tools in biosensing of natural RNA by fluorescence. The background fluorescence of single strands was significantly reduced, while upon formation of complexes with target miR- 7 and circRNA emission increased giving remarkably high quantum yields (O _f up to 0.99) and, therefore, low limit of detection values (< 5 nM in solution) . Importantly, this was achieved by internal modification of the LNA/DNA oligonucleotides providing additional thermal stability and selectivity of the probes towards the target. Accompanied by simple design and robust CuAAC synthetic route, simple purification and high yields of the products, effective target binding, low single-strand background signal (D up to 10.6) and consistent specific fluorescence, the novel probes could become a new platform for biosensing of natural RNA in vitro and in vivo.

The invention also provides a duplex formed between the fluorescent oligonucleotides of the invention, and the complementary DNA or RNA, as well as the use of the fluorescent oligonucleotide according to the invention as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA.

Hybridization of the prepared probes with target DNA and RNA strands was studied using 7 ^~ _m and circular dichroism (CD) experiments in a medium salt phosphate buffer (Table 1, EXAMPLES, Figures 3-4, Table S3) . For all the modified probes S-shape of the melting curves and the characteristic CD signal of an A/B type duplex confirmed successful adoption of the dyes within the duplexes (EXAMPLES, Figures 3-4) . Additional LNA nucleotides improved binding affinities of the modified probes in spite of 5-9 °C destabilizing effect of monomer U ^p (Table 1, 7 ^~ _m for ON 1-ON4 compared to unmodified references) . Perylene-labelled probes P1-P2 showed high binding affinity towards complementary DNA/RNA (7 ^~ _m 64-68 °C), which is most likely caused by additional stacking interactions provided by the polyaromatic hydrocarbons within the double stranded complexes. In turn, xanthene-modified probes P3- P6 showed superior binding affinity towards RNA in comparison to DNA targets, pointing at better adoption of the bulky xanthene fluorophores within the corresponding DNA: RNA hybrids (7 ^" _m 55-57 °C vs. 59-62 °C for the duplexes with DNA and RNA targets,

respectively) . However, incorporation of the fluorophores within monomers M ² -M ³ had a negative affect on 7 ^~ _m values compared to U ^p modified precursors and LNA/DNA references (7 ^~ _m decrease ~ 5 °C) . Finally, all the probes discriminated a single mismatch in central positions of DNA and RNA targets by -3.5-8 °C and -1-8 °C, respectively (EXAMPLES, Figure 4) . Notably, the 7 ^~ _m values were comparable to those for 5'- and internally labeled LNA/DNA fluorescent probes obtained from commercial suppliers (EXAMPLES, Tables S2-S3) . This additionally confirms successful adoption of the bulky dyes within the duplexes formed by the P1-P6. Finally, since all the duplexes having single-nucleotide mismatches were formed at the temperature above 19 °C, all of them were subjected to the fluorescence studies described below.

Fluorescence sensing of hybridization with complementary miRNA, circRNA and their cDNA analogues was studied by in vitro experiments at ambient (19 °C) and elevated temperature (37 °C). The latter was done in order to evaluate RNA biosensing potential of P1-P6 in vivo. The in vitro assay using P1-P6 was characterized by series of important photophysical and diagnostic parameters: discrimination values (D), fluorescence quantum yields (O _f ), fluorescence brightness (FB) and limit of target detection (LOD) values (Table 2; EXAMPLES, Table S4, Figure 6). Perylene-labelled probes P1-P2 displayed remarkably high D values (up to 10.6), accompanied by high fluorescence quantum yields and hence, high FBs at both 19 °C and 37 °C. The probes P3-P4 showed the highest O _f and FB values (up to 0.99 and 138, respectively), also at elevated temperature. Remarkably, high FBs resulted in low LOD values for P1-P4 (< 5-10 nM), which is a strong benefit for diagnostic application of the probes. Finally, probes P5-P6 containing 6-ROX fluorophores showed FB and LOD values comparable to those for P1-P2, although lower D values between single-stranded conjugates and corresponding complexes with miR-7 and circRNA (Table 2).

Table 2 Spectral, photophysical properties and diagnostic characteristics of the complexes between the probes and RNA targets in a medium salt buffer. ³

^a ^ ^abS _m m λ ²¹ , λ^ _^ , <% FB and LOD are maxima of absorbance, excitation wavelength, fluorescence maxima, fluorescence quantum yield, FB (fluorescence brightness) = <i> _f ^χ e _max , and limit of target detection values. <i> _f values are measured by a relative method (EXAMPLES) using perylene and oxazine 170 perchlorate as fluorescence standards. D is determined at 19 °C as a ratio of fluorescence intensities at fluorescence maximum of double-stranded complex to the corresponding single-stranded probe (452 nm for P1-P2, 530 nm (P3-P4), and 605 nm (P5-P6)). LODs were determined by series of target titration experiments described in EXAMPLES. The observed quenching of fluorescence within single-stranded P1-P4 most likely results from aggregation of the hydrophobic dyes surrounded by aqueous media, since both extinction coefficient and ratio of visible absorbance bands I/II of P1-P4 vary for the single strands and duplexes (EXAMPLES, Figure 2) . In turn, as suggested by altered ratio of absorbance bands and increased extinction, hybridization results in positioning of the dyes in a less hydrophilic environment, resulting in the observed light-up of fluorescence.

Next, none of the commercially available 5'- or internally labelled LNA/DNA probes R1-R8 used showed sensitivity of fluorescence to single-nucleotide mismatch in the RNA/cDNA targets (EXAMPLES, Figure 7; R1-R8 contain fluorophores with similar optical properties to M ¹ -^! ³ ). On the contrary, "clickable" probes P1-P6 showed moderate fluorescence mismatch discrimination in cDNA targets (i.e. only numerous positions were discriminated ; data not shown), and efficient discrimination in both miR-7 and circRNA targets by quenching of fluorescence (D 0.1-0.7; EXAMPLES, Tables S4-S5, Figure 7) . This implies that the performed internal incorporation of monomers M*-M ³ with expanded aromatic n-electron systems results in the probes which are highly sensitive to even minor changes in

microenvironment, e.g. to single-nucleotide mismatches.

A critical challenge for biosensing of single-nucleotide polymorphisms (SNPs) in natural RNA targets is that most often a mutant genotype is present at a very low concentration with respect to the wild-type variant. Therefore, as the final aspect, biosensing of the biologically relevant mixtures of a wild-type and mismatched circRNA targets was performed using the selected bright probe P4 and cicrRNA target MT13 containing A→G mismatch in the central position (EXAMPLES, Tables S3,S7) . Already at 0.1 mol % of the mutant RNA fluorescence intensity of P4 was reduced by 33%, whereas 0.3 mol % of MT5 resulted in 40% quenching of the probe's fluorescence. Finally, high base specificity of fluorescence signal is an additional advantage of the prepared probes in genotyping of RNA associated with e.g . cancer or infectious diseases.

The invention also provides simple, effective routes to the fluorescent oligonucleotides described above, and starting materials for the synthesis.

The fluorescent oligonucleotides described above can be synthesised from an oligonucleotide comprising one or more 2'-0-alkylnyl nucleotide units of formula (I) :

(I)

wherein B is a purine or pyrimidine nucleobase and A is a linker moiety. All features of the fluorescent oligonucleotide set out above are also relevant for the oligonucleotide starting materials.

Oligonucleotides comprising one or more 2'-0-propargyl nucleotide units of formula (I) include ON I, ON2, ON3 and ON4, as per Table 1.

The oligonucleotide according to the invention may comprise 2 or more, such as 3 or more, 4 or more, 5 or more, such as e.g . 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-0-alkylnyl nucleotide units of formula (I) .

Starting from a oligonucleotide comprising one or more 2'-0-alkylnyl nucleotide units of formula (I), the fluorescent oligonucleotides of the invention can be synthesised . Synthesis comprises reacting the oligonucleotide according to the invention 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 each 2'-0-alkylnyl nucleotide unit 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.

CuAAC click conjugation of ON 1-ON2 with azides 3-5 was performed either under microwave conditions for 15 min at 60 °C (azide 3), or at ambient temperature for 24 h (azides 4-5), giving the desired products P1-P6 in high purity and yields of 74-83%

(EXAMPLES) . Microwave conditions of the click reactions were applied for incorporation of monomer M ¹ in order to improve solubility of the azide 3. The products were characterized by IE HPLC and MALDI MS (EXAMPLES, Table SI) . The present invention also provides a method for designing and synthesising a fluorescent oligonucleotide probe for a DNA or RNA sequence to be detected, said fluorescent oligonucleotide probe comprising two or more fluorescent 2'-0-substituted nucleotide monomers of formula (II), said method comprising the steps of: providing an oligonucleotide comprising two or more 2'-0-alkylnyl nucleotide units of formula (I), located internally in said oligonucleotide, said oligonucleotide having a DNA or RNA sequence complementary to the DNA or RNA sequence to be detected, using one or more of the following design rules:

a. the oligonucleotide has a total length of between 10 and 30 nucleotides,

preferably between 13 and 23 nucleotides;

b. adjacent 2'-0-alkylnyl nucleotide units of formula (I) are separated by 1 or 2 nucleotides A, C or T;

c. the oligonucleotide has one or more locked nucleic acid (LNA) units, such as two or more LNA units, or three or more LNA units, wherein said two or more locked nucleic acid (LNA) units are separated by at least one other nucleotide A, C, T or G, and said one or more LNA units are not located between two 2'- O-alkylnyl nucleotide units of formula (I) in said oligonucleotide; reacting the oligonucleotide from step i. at said two or more 2'-0-alkylnyl nucleotide units with fluorescent dye compound having the structure FL-N ₃ , in which FL constitutes a fluorescent moiety and N3 constitutes an azide moiety wherein said reaction is a copper (I)-catalyzed azide alkyne cycloaddition, thereby incorporating two or more fluorescent moieties FL into said oligonucleotide.

EXAMPLES

Materials and methods. Reagents obtained from commercial suppliers were used as received. Reagents and solvents for click chemistry were obtained from commercial suppliers (Fluka, Lumiprobe, Sigma-Aldrich), and were used as received; tris[(l-benzyl-lH-l,2,3- triazol-4-yl)methyl] amine (TBTA) was prepared following published procedures (7 ^" . R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin ,Org. Lett, 2004, 6, 2853). Stock solutions for click chemistry were prepared as described in recent publications (A. V. Ustinov, I. A. Stepanova, V. V. Dubnyakova, T. S. Zatsepin, E. V. Nozhevnikova, V. A. Korshun, Russ. J. Bioorg.

Chem. , 2010, 36, 401. And A.H. El-Sagheer, T. Brown, Chem. Soc. Rev., 2010, 39, 1388. c) F. Amblard, J. H. Cho, R. F. Schinazi, Chem. Rev., 2009, 109, 4207) Click reactions were performed in 2 mL reactor tubes under argon and vigorous stirring in a microwave reactor (Emrys Creator, Personal Chemistry), or in 2 mL eppendorf tubes at ambient temperature. Perylene and ozaxine 170 perchlorate were used as standards for emission quantum yield measurements after recrystallization. Photochemical studies were performed using spectroquality methanol and cyclohexane. Other solvents and reagents applied in this study were used as received .

Synthesis and purification of modified oligonucleotides. LNA phosphoramidites (G ^L , A ^L and C ^L ) and phosphoramidite 1 were obtained from commercial suppliers (Exiqon and Jena Bioscience, respectively), and were incorporated into synthetic oligonucleotides following manufactures' protocols. Oligonucleotide synthesis was carried out on AKTA oligopilot plus instrument (GE Healthcare Life Sciences) in 1000 nmol scale using standard manufacturer's protocols. Coupling yields based on the absorbance of the dimethoxytrityl cation released after each coupling were approximately 98-99% for modified monomers U ^p , LNA and unmodified DNA phosphoramidites. Cleavage from solid support and removal of nucleobase protecting groups were performed using standard conditions (32% aqueous ammonia for 12 h at 55 °C) . The modified oligonucleotides were purified by DMT-OFF RP-HPLC using the Waters Prep LC 4000 equipped with Xterra MS C18-column (10 μιη, 300 mm x 7.8 mm) . Elution was performed starting with an isocratic hold of A-buffer for 2 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 precipitation (acetone, -18 °C, 12 h) and washing with acetone (2 ^χ 0.5 mL) . The identity and purity of the products were verified by MALDI-TOF mass spectrometry (Ultraflex II, Bruker) and IE HPLC, respectively. IE 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 LiCI0 ₄ , C-buffer: 250 mM 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 strands were obtained from commercial suppliers and used without further purification.

General method for microwave-assisted CuAAC reactions. Starting oligonucleotide ON 1-ON2 (30 nmol) was dissolved in fresh MQ water (325 μΙ_) in a microwave-tube. DMSO (435 μΙ_), 2 M triethylammonium acetate buffer (pH 7.4; 100 μΙ_), azide 3 (40 μί of 10 mM solution in DMSO), ascorbic acid (10 μΙ_ of 50 mM freshly prepared stock solution) and Cu(II)- TBTA equimolar complex (50 μL of 10 mM stock solution) were subsequently added. The resulting mixture was 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 twice through Illustra NAP-10 column (GE Healthcare) following manufacture's protocol . The resulting solution was evaporated; the resulting conjugates P1-P2 were analyzed by MALDI TOF mass spectrometry and IE HPLC (Table S2, Figures 2- 3). Yields: 74% (PI), 78% (P2).

General method for CuAAC reactions with azides 4-5. Starting oligonucleotide ON1- ON2 (30 nmol) was dissolved in fresh MQ water (525 μΙ_) in 2 mL eppendorf tube. DMSO (235 μΙ_), 2 M triethylammonium acetate buffer (pH 7.4; 100 μΙ_), corresponding azide (4-5; 40 μΙ_ of 10 mM solution in DMSO), ascorbic acid (10 μΙ_ of 50 mM freshly prepared stock solution) and Cu(II)-TBTA equimolar complex (50 μΙ_ of 10 mM stock solution) were subsequently added. The resulting mixture was tightly closed, mixed on vortex and kept at room temperature for 24 h. The reaction was afterwards filtrated twice through Illustra NAP- 10 column (GE Healthcare) following manufacture's protocol. The resulting solution was evaporated; the resulting conjugates P3-P6 were analyzed by MALDI TOF mass

spectrometry and IE HPLC (Table S2, Figures 2-3). Yields: 81% (P3), 77% (P4), 79% (P5), 83% (P6).

Table SI. IE HPLC retention times and MALDI MS of modified oligonucleotides.

# Sequence, 5'→3' Ret. time, Found m/z Calc. m/z min

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

ONI d(ACAACAAA ^L ATC ^L ACU ^p AGU ^p CU ^p TCC ^L A) 8 ^~ 1M 7172 7173

ON2 d ( G A AG AC ^L GTG ^L G A U ^P TU ^P TC U ^P G G A ^L AG A) 8.94 7382 7382

ON3 d(ACAACAAA ^L ATC ^L ACTAGTCTTCC ^L A) 8.94 7053 7056

ON4 d(GAAGAC ^L GTG ^L GATTTTCTGGA ^L AGA) 8.94 7265 7265

PI d(ACAACAAA ^L ATC ^L ACM ¹ AGM ¹ CM ¹ TCC ^L A) 10.78 8179 8178

P2 d(GAAGAC ^L GTG ^L GAM ¹ TM ¹ TCM ¹ GGA ^L AGA) 11.96 8392 8387

P3 d(ACAACAAA ^L ATC ^L ACM ² AGM ² CM ² TCC ^L A) 9.87 8540 8542.5 d(GAAGAC ^L GTG ^L GAM ² TM ² TCM ² GGA ^L AGA) 8751 8751.5 d(ACAACAAA ^L ATC ^L ACM AGM CM TCC ^L A) 10.17 9019 9024 d(GAAGAC ^L GTG ^L GAM TM TCM GGA ^L AGA) 9230 9233

UV-visible absorbance and thermal denaturation studies were performed in a medium salt phosphate buffer (100 mM sodium chloride, 10 mM sodium phosphate, 0.1 mM EDTA, pH 7.0) on a Beckman Coulter DU800 UV/VIS Spectrophotometer equipped with Beckman Coulter High Performance Temperature Controller. Concentrations of oligonucleotides were calculated using the nearest-neighbour method and the following extinction coefficients (OD ₂₅₀ / mol) : G, 10.5; A, 13.9; T/U/U ^p , 7.9; C, 6.6; M ¹ , 33.2; M ² ; 11.8; M ³ , 20.0;

Reference fluorophores: iFT, 6FAM, 6.5; 5HEX, 17.2; AlexaFluor 488, 21.3. The data for absorbances of iFT, 6FAM, 5HEX and AlexaFluor 488 was obtained from commercial suppliers. The complementary strands were thoroughly mixed, denatured by heating to 85 °C for 10 min and subsequently cooled overnight to the starting temperature of spectroscopic experiment. Thermal denaturation temperatures (7 ^~ _m values, °C) were determined as the maxima of the first derivative of the thermal denaturation curve (A ₂ &o vs. temperature). Reported 7 ^~ _m values are an average of two measurements within ± 0.5 °C.

CD measurements

CD spectra were recorded on JASCO J-815 CD Spectrometer equipped with CDF 4265/15 temperature controller. Samples for CD measurements were prepared as described in the thermal denaturation studies section except that a concentration of 2.0 μΜ of both the complementary strands was used. Quartz optical cells with a path-length of 0.5 cm were used.

Table S2. Reference probes and mismatched miR-7 targets used in this study.

# Sequence Supplier

Rl 5'-d(ACAACAAA ^L ATC ^L AC(iFT)AG(iFT)C(iFT)TCC ^L A) Exiqon - ( 6 F AM ) -d ( ACAACAAA ^L ATC ^L ACTAGTCTTCC ^L A) Exiqon

R3 5'-(5HEX)-d(ACAACAAA ^L ATC ^L ACTAGTCTTCC ^L A) Exiqon

5'-d(GAAGAC ^L GTG ^L GA(AF)T(AF)TC(AF)GGA ^L AGA) miR-7 5'-r(UGG AAG ACU AGU GAU UUU GUU GU) IDT

MT1 5'-r(UGG AAG ACA AGU GAU UUU GUU GU) IDT

MT2 5'-r(UGG AAG ACG AGU GAU UUU GUU GU) IDT

MT3 5'-r(UGG AAG ACC AGU GAU UUU GUU GU) IDT

MT4 5'-r(UGG AAG ACU UGU GAU UUU GUU GU) IDT

MT5 5'-r(UGG AAG ACU GGU GAU UUU GUU GU) IDT

MT6 5'-r(UGG AAG ACU CGU GAU UUU GUU GU) IDT

MT7 5'-r(UGG AAG ACU AGU GAU UGU GUU GU) IDT

MT8 5'-r(UGG AAG ACU AGU GAU UAU GUU GU) IDT

* A mismatched nucleotide is shown in red. ** AF = AlexaFluor 488; the corresponding azide obtained from Glen Research was used in click chemistry reaction with ONI according to the similar procedure as described for 4-5. For the chemical structures of the modifications, see web-sources of the corresponding suppliers.

Table S3. Reference probes and mismatched circRNA targets used in this study.

# Sequence Supplier R5 5'-d(GAAGAC ^L GTG ^L GA(iFT)T(iFT)TC(iFT)GGA ^L AGA) Exiqon

R6 5'-(6FAM)-d(GAAGAC ^L GTG ^L GATTTTCTGGA ^L AGA) Exiqon

R7 5'-(5HEX)-d(GAAGAC ^L GTG ^L GATTTTCTGGA ^L AGA) Exiqon

R8** 5'-d(GAAGAC ^L GTG ^L GA(AF)T(AF)TC(AF)GGA ^L AGA) Glen Res.** circRNA 5'-r(UCU UCC AGA AAA UCC ACG UCU UC) IDT

MT9 5'-r(UCU UCC AGA UAA UCC ACG UCU UC) IDT

MTIO 5'-r(UCU UCC AGA GAA UCC ACG UCU UC) IDT

MT11 5'-r(UCU UCC AGA CAA UCC ACG UCU UC) IDT

MT12 5'-r(UCU UCC AGA AUA UCC ACG UCU UC) IDT

MT13 5'-r(UCU UCC AGA AGA UCC ACG UCU UC) IDT

MT14 5'-r(UCU UCC AGA ACA UCC ACG UCU UC) IDT

MT15 5'-r(UCU UCC AGA AAA UCC AGG UCU UC) IDT

MT16 5'-r(UCU UCC AGA AAA UCC AUG UCU UC) IDT

* A mismatched nucleotide is shown in red. ** AF = AlexaFluor 488; the corresponding azide obtained from Glen Research was used in click chemistry reaction with ON2 according to the similar procedure as described for 4-5. For the chemical structures of the modifications, see web-sources of the corresponding suppliers.

Fluorescence steady-state emission and excitation studies. Determination of quantum yield and limit of target detection values. Fluorescence spectra were obtained using a PerkinElmer LS 55 luminescence spectrometer equipped with a Peltier temperature controller using the following excitation/emission wavelengths: 425/452 nm (P1-P2)

480/530 nm (P3-P4), 580/605 nm (P5-P6), 494/520 nm (R1-R2, R5-R6), 533/550 nm (R3, R7), and 494/519 nm (R4,R8) .

All the measurements was performed at excitation slit of 4.0 nm, emission slit of 2.5 nm, scan speed of 120 nm/min and 0.25-1.0 μΜ concentrations of the single-stranded probe or the corresponding complementary complex in a medium salt buffer described above. The fluorescence quantum yields (O _f ) were measured by the relative method using standards of highly diluted solutions of perylene (O _f = 0.93) and oxazine 170 perchlorate (O _f = 0.58) in cyclohexane and methanol, respectively. The samples used in quantum yield measurements were not degassed ; concentrations were 0.25 μΜ.

Limit of detection (LOD) values were determined by series of dilution experiments following previously described protocol. ¹ Relevant fluorescent oligonucleotides at concentrations 500, 250, 100, 50, 20, 10 and 5 nM were mixed in a medium salt phosphate buffer in molar ratios described above. Upon annealing, a fluorescence signal was measured at λ" 452 nm (P1-P2), 530 nm (P3-P4), and 605 nm (P5-P6) . LOD value of each complex was then defined as a lowest complex concentration such that the fluorescence signal to noice ratio (S/N) relative to the blank solution of a medium salt phosphate buffer was minimum three.

Table S4. Spectroscopic and photophysical properties of modified oligonucleotides and duplexes. ³

^abs _max bands I, II abs

OF ( 19 °C/37 °C) FB at 19 °C

(nm) (nm)

P#

Duplex with Duplex with Duplex with Duplex with complimentary complimentary complimentary complimentary

SSP SSP SSP SSP

DNA RNA DNA RNA DNA RNA DNA RNA

423 423 423 457 457 459 0.07/ 0.31/ 0.58/

PI 3 33 27

451 450 451 487 487 490 0.05 0.22 0.45

423 423 423 457 458 458 0.17/ 0.27/ 0.47/

P2 9 34 34

450 450 451 489 488 488 0.09 0.24 0.40

0.39/ 0.96/ 0.90/

P3 507 506 506 531 525 526 68 138 102

0.51 1.00 0.96

0.17/ 0.58/ 0.90/

P4 511 509 506 531 530 528 27 85 133

0.25 0.62 0.99

547 549 549 0.26/ 0.16/ 0.23/

P5 605 605 605 41 37 32

584 584 584 0.26 0.12 0.23

547 546 546 0.30/ 0.17/ 0.18/

P6 607 607 607 42 21 30

589 585 585 0.23 0.15 0.18 [a] SSP = single-stranded probe; λ _max , λ _max and O _f are absorbance, fluorescence maxima and fluorescence quantum yield, respectively. Fluorescence brightness (FB) values were calculated using equation : FB = 8max x OF, where 8 _ma x is the maximal molar extinction coefficient of the probe and OF is the corresponding fluorescence quantum yield.

Table S5. Representative discrimination factors for the probes and references sensing circRNA targets containing single-nucleotide mismatches. ³

Target

^~ ΡΪ P3 P5 Rl R2 R3 R4 ^{^}

MT1 67Ϊ δ (λ2 θΤδ θ 5 (λ8

MT2 0.5 0.4 0.2 1.2 0.9 1.3 0.7

MT3 0.6 0.9 0.4 1.4 0.9 1.4 0.8

MT4 0.3 0.4 0.3 1.2 0.5 0.9 0.8

MT5 0.4 0.5 0.1 1.5 0.9 1.4 0.7

MT6 0.5 0.7 0.1 1.3 0.9 1.2 1.2

MT7 1.0 0.6 0.2 1.2 1.0 1.2 1.2

MT8 0.8 1.5 0.4 1.5 1.0 1.2 1.3 ^a D is determined at 19 °C as a ratio of fluorescence intensities at fluorescence maximum of fully complementary double-stranded complex to the corresponding single-mismatched complex. Table S6. Representative discrimination factors for the probes and references sensing circRNA targets containing single-nucleotide mismatches. ³

Probe* and D

RNA

Target

PI P3 P5 R5 R6 R7 R8

MT9 0.3 0.5 0.7 2.1 1.0 1.0 1.2

MT10 0.6 0.9 0.6 0.6 1.1 0.6 1.5

MT11 0.1 0.5 0.5 1.5 1.3 1.0 1.0

MT12 0.2 0.8 0.3 1.3 0.9 1.0 1.1

MT13 0.5 0.5 0.3 0.8 1.2 0.5 1.1

MT14 0.3 0.4 0.5 1.5 0.9 0.7 1.1

MT15 0.2 0.4 0.6 0.9 1.4 1.0 1.1

MT16 0.2 0.7 0.4 0.9 1.1 1.0 1.3 ^a D is determined at 19 °C as a ratio of fluorescence intensities at fluorescence maximum of fully complementary double-stranded complex to the corresponding single-mismatched complex. Table S7. Fluorescence sensing of the pure fully complementary circRNA, single-mismatched target MT13, and of their mixtures by the probe P4. ^a

Quenching of

fluorescence in

% Mutant presence of mutation

% Wild-type Fluorescence

target

target (mol) intensity

(mol) (%)

(single-stranded

probe)

100.0 0.0 16 100

66.7 33.3 15 74

16.1 83.9 20 65

4.0 96.0 25 57

1.0 99.0 25 57

0.3 99.8 33 40

0.1 99.9 44 33

0.0 100.0 57 0 ^a Total concentration of the probe and RNA was 1.0 μΜ ; excitation/emission wavelengths: 480/525 nm. The following numbered aspects of the invention are provided :

Aspect 1. A fluorescent oligonucleotide, comprising two or more fluorescent 2'-0- substituted nucleotide monomers of formula (II) located internally in said oligonucleotide:

(Π) wherein B is a purine or pyrimidine nucleobase, A is a linker moiety and wherein each FL constitutes the same fluorescent moiety.

Aspect 2. The fluorescent oligonucleotide according to aspect 1, wherein B is a pyrimidine nucleobase.

Aspect 3. The fluorescent oligonucleotide according to any one of aspects 1-2, comprising two or more FL moieties, each being the same cyanine, perylene, pyrene or other PAH, or xanthene fluorescent moiety.

Aspect 4. The fluorescent oligonucleotide according to any one of aspects 1-3 comprising two 2'-0-substituted nucleotide monomers of formula (II) .

Aspect 5. The fluorescent oligonucleotide according to one of aspects 1-3 comprising three 2'-0-substituted nucleotide monomers of formula (II) .

Aspect 6. The fluorescent oligonucleotide according to any one of aspects 1-5 having a total length of between 10 and 30 nucleotides, preferably between 13 and 23 nucleotides.

Aspect 7. The fluorescent oligonucleotide according to any one of aspects 1-6, wherein adjacent 2'-0-substituted nucleotide monomers of formula (II) are separated by 1 or 2 nucleotides A, C or T.

Aspect 8. The fluorescent oligonucleotide according to any one of aspects 1-7 comprising one or more locked nucleic acid (LNA) units, such as two or more LNA units, or three or more LNA units. Aspect 9. The fluorescent oligonucleotide according to aspect 8, wherein said two or more locked nucleic acid (LNA) units are separated by at least one other nucleotide A, C, T or G.

Aspect 10. The fluorescent oligonucleotide according to any one of aspects 8-9, wherein said one or more LNA units are not located between two 2'-0-substituted nucleotide moieties of formula (II) in said oligonucleotide.

Aspect 11. The fluorescent oligonucleotide according to any one of aspects 1-10, comprising sequence P1-P6 as described herein.

Aspect 12. A duplex formed between the fluorescent oligonucleotide according to any one of aspects 1-11, and the complementary DNA or RNA.

Aspect 13. Use of the fluorescent oligonucleotide according to any one of aspects 1-12 as a fluorescent LNA/DNA probe for the detection of complementary DNA and/or RNA.

Aspect 14. Use of the fluorescent oligonucleotide according to any one of aspects 1-11, or the duplex according to aspect 12, for the detection of an autoimmune antibody.

Aspect 15. An oligonucleotide comprising one or more 2'-0-alkylnyl nucleotide units of formula (I) located internally in said oligonucleotide:

(I) wherein B is a purine or pyrimidine nucleobase and A is a linker moiety.

Aspect 16. The oligonucleotide according to aspect 15, wherein B is a pyrimidine nucleobase. Aspect 17. The oligonucleotide according to any one of aspects 15-16, comprising sequence ON1-ON4, as described herein.

Aspect 18. The oligonucleotide according to any one of aspects 15-17 comprising 2 or more, such as 3 or more, 4 or more, 5 or more, such as e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-0- alkylnyl nucleotide units of formula (I) .

Aspect 19. The oligonucleotide according to aspect 18 comprising two 2'-0- alkylnyl nucleotide units of formula (I) .

Aspect 20. The oligonucleotide according to aspect 18 comprising three 2'-0- alkylnyl nucleotide units of formula (I) .

Aspect 21. The oligonucleotide according to any one of aspects 15-20 having a total length of between 10 and 30 nucleotides, preferably between 13 and 23 nucleotides

Aspect 22. The oligonucleotide according to any one of aspects 18-21 wherein adjacent 2'- O-alkylnyl nucleotide units of formula (I) are separated by 1 or 2 nucleotides A, C or T.

Aspect 23. The oligonucleotide according to any one of aspects 15-22 comprising one or more locked nucleic acid (LNA) units, such as two or more LNA units, or three or more LNA units.

Aspect 24. The oligonucleotide according to aspect 23, wherein said two or more locked nucleic acid (LNA) units are separated by at least one other nucleotide A, C, T or G.

Aspect 25. The oligonucleotide according to any one of aspects 23-24, wherein said one or more LNA units are not located between two 2'-0-alkylnyl nucleotide units of formula (I) in said oligonucleotide.

Aspect 26. A method for synthesizing a fluorescent oligonucleotide, said method comprising the step of reacting the oligonucleotide according to any one of aspects 18-25 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 each 2'-0-alkylnyl nucleotide unit 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 27. A method for designing and synthesising a fluorescent oligonucleotide probe for a DNA or RNA sequence to be detected, said fluorescent oligonucleotide probe comprising two or more fluorescent 2'-0-substituted nucleotide monomers of formula (II) located internally in said oligonucleotide, said method comprising the steps of: i. providing an oligonucleotide comprising two or more 2'-0-alkylnyl nucleotide units of formula (I), herein located internally in said oligonucleotide, said

oligonucleotide having a DNA or RNA sequence complementary to the DNA or RNA sequence to be detected, using one or more of the following design rules: a. the oligonucleotide has a total length of between 10 and 30 nucleotides,

preferably between 13 and 23 nucleotides; b. adjacent 2'-0-alkylnyl nucleotide units of formula (I) are separated by 1 or 2 nucleotides A, C or T; c. the oligonucleotide has one or more locked nucleic acid (LNA) units, such as two or more LNA units, or three or more LNA units, wherein said two or more locked nucleic acid (LNA) units are separated by at least one other nucleotide A, C, T or G, and said one or more LNA units are not located between two 2'- O-alkylnyl nucleotide units of formula (I) in said oligonucleotide; ii. reacting the oligonucleotide from step i. at said two or more 2'-0-alkylnyl

nucleotide units with 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, thereby incorporating two or more fluorescent moieties FL into said oligonucleotide.