tection of natural RNA using rationally designed “ clickable ” oligonucleotide probes †

Interdisciplinary Nanoscience Center (iNAN and Genetics, Aarhus University, Gustav W E-mail: jk@mb.au.dk; Tel: +45 28992086 Nucleic Acid Center, Department of Physics Southern Denmark, DK-5230 Odense M, Denma † Electronic supplementary information purication and characterization of the limit of target detection calculations, an targeting complementary and singly-mis 10.1039/c4ra07165d Cite this: RSC Adv., 2014, 4, 45653

RNA targeting is a rapidly evolving eld of research and clinical diagnostics which applies synthetic uorescent probes and modern biophysical techniques to monitor desired targets in vitro and in vivo. 1 Preparation of uorescent oligonucleotides with high potential in RNA detection by a simple and rapid method, e.g. by uorescence, is an important aspect of current nucleic acid chemistry.Recently copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) click chemistry has been successfully applied for nucleic acid labelling, allowing preparation of various oligonucleotides in high yields and purity. 2,3urthermore, attachment of uorophores by click chemistry to 2 0 -O-propargyl uridine scaffold U P was proven to yield probes with promising properties in diagnostics (Fig. 1). 4 However, in order to further utilize "clickable" oligonucleotides in diverse analytical experiments, the uorescent probes have to meet several additional requirements, such as high photo-and chemical stability, low uorescence background signal from single stranded molecules, high binding specicity and low limit of target detection. 5Ideally, incorporation of uorescent dyes should furthermore be simple and allow high exibility in probe design.
MicroRNAs (miRNAs) are short ($22 nucleotides) noncoding RNA molecules that post-transcriptionally repress the expression of protein-coding genes by binding to 3 0 -untranslated regions of the target mRNAs. 6An increasing amount of evidence shows that miRNAs control a large number of biological processes, including cell differentiation, tumourigenesis and regulation of metabolism.A thoroughly studied miRNA is miRNA-7 (miR-7) which is highly expressed in brain neurons and also an important negative regulator of oncogenes and, hence, functioning as a tumor suppressor factor in oncogenesis. 7miR-7 was recently identied to be selectively expressed within the hypothalamus, a part of the brain that controls vital bodily functions. 7nother class of recently discovered RNA molecules with important, yet not fully studied, regulatory functions are circular RNAs (circRNAs). 8,9Interestingly, it was found that a human circRNA, antisense to the cerebellar degenerationrelated protein 1 transcript (CDR1as), is densely bound by miR-7 molecules in cells and presumably acts as a regulator of miR-7 activity. 97][8][9] In the present work we aimed at a robust approach to preparation of uorescent probes with high binding affinity and efficient uorescence detection of miR-7 and circRNA (Fig. 1).Herein, the design and synthesis of miR-7 and circRNA targeting oligonucleotides containing 2 0 -Opropargyl uridine scaffolds U P , their uorescent labelling by CuAAC click reactions, and spectroscopic characterization of the resulting uorescent probes in vitro are reported.][8][9] First, we selected perylene and two rhodamine derivatives, 5-R110 and 6-ROX, as uorophores with interesting optical properties to be internally attached to the scaffold U P within new probes (Fig. 1 and Scheme 1). 4,10,11Second, three incorporations of the dyes within 13-21mer oligonucleotides were previously demonstrated to be optimal for spectral properties of the probes. 47][8][9] Third, the "clickable" scaffolds U P within ON1, ON2 were separated by 1-2 nucleotides in order to achieve optimal uorescence emitted from the probes upon target binding. 4As a nal design aspect, additional LNA (locked nucleic acids; Scheme 1) monomers were incorporated into ON1, ON2 to enhance binding affinity and speci-city of the probes to complementary targets. 11Finally, reference probes ON3, ON4 were prepared in order to evaluate inuence of the modied monomers U P and M 1 -M 3 on target binding (Table 1).
CuAAC click conjugation of ON1, 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 and 5), giving the desired products P1-P6 in high purity and yields of 74-83% (ESI †).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 ion-exchange (IE) HPLC and MALDI MS (ESI, Table S1 †).Incorporation of the uorescent monomers into ON1, ON2 was further conrmed by characteristic bands in the visible region of the corresponding absorbance spectra (Table 2; ESI, Table S4 and Fig. S1 †).Notably, hybridization had an inuence on the shape and intensity of absorbance curves which implied interaction of the dyes with microenvironment, e.g.dye aggregation and stacking interactions between the dyes and nucleobases. 4,12,13ybridization of the prepared probes with target DNA and RNA strands was studied using T m and circular dichroism (CD) experiments in a medium salt phosphate buffer (Table 1, ESI, Fig. S2, S3 and Table S3 †).For all the modied probes S-shape of the melting curves and the characteristic CD signal of an A/B type duplex conrmed successful adoption of the dyes within the duplexes (ESI, Fig. S2 and S3 †). 11Additional LNA nucleotides improved binding affinities of the modied probes in spite of 5-9 C destabilizing effect of monomer U P (Table 1, T m for ON1-ON4 compared to unmodied references).Perylenelabelled probes P1, P2 showed high binding affinity towards complementary DNA/RNA (T m 64-68 C), which is most likely caused by additional stacking interactions provided by the polyaromatic hydrocarbons within the double stranded complexes. 10Furthermore, rhodamine-modied probes P3-P6 showed superior binding affinity towards RNA in comparison to DNA targets, indicating at better adoption of the bulky rhodamine uorophores within the corresponding DNA-RNA hybrids (T m 55-57 C vs. 59-62 C for the duplexes with DNA and RNA targets, respectively).However, incorporation of the uorophores within monomers M 2 , M 3 had a negative effect on T m values compared to U P -modied and LNA/DNA references (T m decrease $ 5 C).All the probes discriminated a single mismatch in the central positions of DNA and RNA targets by À3.5 to 8 C and À1 to 8 C, respectively (ESI, Fig. S3 †).Notably, the T m values were comparable to those for 5 0 -and internally labeled LNA/DNA uorescent probes obtained from commercial suppliers (ESI, Tables S2 and S3 †).Finally, since all the duplexes having single-nucleotide mismatches were formed at the temperature above 19 C, all of them were subjected to the uorescence studies described below.
Fluorescence detection 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 potential of P1-P6 for in vivo applications.The in vitro assay using P1-P6 was characterized by series of photophysical and diagnostic parameters: discrimination values (D), uorescence quantum yields (F f ), uorescence brightness (FB) and limit of target detection (LOD) values (Table 2; ESI, Table S4 and Fig. S4 †) 10 In order to efficiently detect a target in vivo, D value of $10 is preferred.Perylene-labelled probes P1, P2 displayed high D values (up to 10.6), accompanied by high uorescence quantum yields and high FBs at both 19 C and 37 C.The probes P3, P4 showed the highest F f and FB values (up to 0.99 and 138, respectively), also at elevated temperature.High FBs resulted in low LOD values for P1-P4 (<5-10 nM), which is benecial for diagnostic applications of the probes. 5However, low D values make them not applicable for diagnostic settings.Finally, P5, P6 showed similar FB and LOD values compared to those observed for P1, P2, and lower D values between single-stranded conjugates and corresponding complexes with miR-7 and circRNA (Table 2).The observed quenching of uorescence 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 (ESI, Fig. S1 †). 13 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 uorescent signal.
Next, none of the commercially available 5 0 -or internally labeled LNA/DNA probes R1-R8 used in this study showed sensitivity of uorescence to single-nucleotide mismatch in the RNA/cDNA targets (D 0.6-1.5;ESI, Fig. S5; † R1-R8 contain uorophores with similar optical properties to M 1 -M 3 ).On the contrary, "clickable" probes P1-P6 showed moderate uorescence mismatch discrimination in cDNA targets (i.e.only numerous positions were discriminated; data not shown), and efficient discrimiation in most of the examined miR-7 and circRNA targets by quenching of uorescence (e.g.D 0.1-0.7 for MT1-MT8 and probes P1, P3, P5; ESI, Tables S4, S5 and Fig. S5 †).Overall, four duplexes out of forty eight studied showed low mismatch discrimination (D 0.9-1.6 by P4: MT1/ MT3/MT8 and P2: MT7; ESI, Table S4 †).This implies that the performed internal incorporation of monomers M 1 -M 3 with expanded aromatic p-electron systems results in the probes which are in general potent to sense minor changes in microenvironment such as single-nucleotide mismatches.
A critical challenge for detecting single-nucleotide polymorphisms (SNPs) in natural RNA targets is that most oen a mutant genotype is present at a very low concentration with respect to the wild-type variant. 14Therefore, as the nal aspect, biologically relevant mixtures of a wild-type and mismatched circRNA targets were analysed using the selected bright probe P4 and circRNA target MT13 containing A / G mismatch in the central position (Fig. 2; ESI, Tables S3 and  S7 †).Fluorescence of fully matched duplex P4-MT13 consistently decreased upon increasing concentration of the a l abs max , l ex , l  max , F f , FB and LOD are maxima of absorbance, excitation wavelength, uorescence maxima, uorescence quantum yield, FB (uorescence brightness) ¼ F f Â 3 max /1000, and limit of target detection values.F f values are measured by a relative method (ESI) using perylene and oxazine 170 perchlorate as uorescence standards.D is determined at 19 C as a ratio of uorescence intensities at uorescence 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 ESI.

Conclusions
In this work the rationally designed "clickable" probes are demonstrated to be promising tools in RNA detection by uorescence.The background uorescence of single strands was signicantly reduced, while upon formation of complexes with target miR-7 and circRNA emission increased giving remarkably high quantum yields (F f up to 0.99) and, therefore, low limit of detection values (<5 nM in solution).Importantly, this was achieved by internal modication 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 purication and high yields of the products, effective target binding, low single-strand background signal (D up to 10.6) and consistent specic uorescence, the "clickable" probes could become a reliable platform for detection of natural RNA.

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

Table 1
14ermal denaturation temperatures of the modified duplexes in a medium salt phosphate buffer a mismatched complex which allows accurate estimation of the mismatch abundance in the corresponding analyte.Finally, high base specicity of uorescence signal is an additional advantage of the prepared probes in genotyping of natural RNA.14