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LNA-modified oligonucleotides are highly efficient as FISH probes Article  in  Cytogenetic and Genome Research · February 2004 DOI: 10.1159/000079569 · Source: PubMed

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Original Article Cytogenet Genome Res 107:32–37 (2004) DOI: 10.1159/000079569

LNA-modified oligonucleotides are highly efficient as FISH probes A. Silahtaroglu,a H. Pfundheller,b A. Koshkin,b N. Tommerupa and S. Kauppinenc a Wilhelm

Johannsen Centre for Functional Genome Research, Dept. of Medical Genetics, Inst. of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen; b Department of Chemistry, c Department of Functional Genomics, Exiqon, Vedbaek (Denmark)

Abstract. Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their ther-

mal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different humanspecific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.

LNA is a bicyclic RNA analog (Fig. 1a) in which the ribofuranose ring in the sugar-phosphate backbone is structurally constrained by a methylene bridge between the 2)-oxygen and the 4)-carbon atoms resulting in a locked 3)-endo conformation, thereby reducing its conformational flexibility and increasing the local organization of the phosphate backbone (Koshkin et al., 1998). The fixed N-type (3)-endo) conformation of the LNA nucleoside, characteristic for A-type double helices, has recently been confirmed by X-ray crystallography and NMR spectroscopy (Obika et al., 1997). In heteroduplexes between LNA oli-

gonucleotides and their complementary DNA oligonucleotides, an overall shift of the duplex structure from a B-like helix towards an A-type helix has been reported. This, in turn, results in higher thermal stability of the LNA-DNA heteroduplexes. Interestingly, LNA monomers are also able to twist the conformation of neighboring DNA nucleotides from an S-type towards an N-type conformation in LNA/DNA mixmer oligonucleotides (Fig. 1b; Bondensgaard et al., 2000; Petersen et al., 2000). LNA obeys the Watson-Crick base-pairing rules with the ability to increase the melting temperature (Tm) of a DNA oligonucleotide probe by 1 to 3 ° C per LNA modification. The unprecedented thermal stability of LNA-DNA heteroduplexes and the high discriminatory power between matched and mismatched target sequences make LNA probes well suited for nucleic acid hybridization-based applications (Jacobsen et al., 2002a, b). Because LNA chemistry is fully compatible with conventional DNA phosphoramidite chemistry, LNA-substituted DNA oligonucleotides can be designed for optimal performance. Furthermore, the electrostatic properties of LNA probes are similar to DNA and RNA oligonucleotides (Fig. 1),

Supported by Danish Research Agency (Project no: 2013-01-0033). Wilhelm Johannsen Centre for Functional Genome Research is established by the Danish National Research Foundation. Received 29 April 2004; revision accepted 28 May 2004. Request reprints from: Asli N. Silahtaroglu Wilhelm Johannsen Centre for Functional Genome Research Department of Medical Genetics, IMBG The Panum Institute, University of Copenhagen Blegdamsvej 3, 2200 N, Copenhagen (Denmark) telephone: (+45) 35 32 78 44; fax: (+45) 35 32 78 45 e-mail: [email protected]

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Fax + 41 61 306 12 34 E-mail [email protected] www.karger.com

© 2004 S. Karger AG, Basel 0301–0171/04/1072–0032$21.00/0

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Fig. 1. (a) Structure of LNA, DNA and RNA. (b) The fixed N-type (C3)-endo) conformation of the LNA nucleotide.

which makes it easy to synthesize LNA oligonucleotide probes using standard phosphoramidite chemistry and oligonucleotide purification methods. Finally, an important practical advantage is that LNA-modified oligonucleotides are fully soluble in water, which makes their handling and use in nucleic acid hybridization experiments simple (Koshkin et al., 1998; Braasch and Corey, 2001). LNA oligonucleotides have been successfully employed in several highly accurate genotyping assays, including genotyping of the apolipoprotein B R3500G SNP (Jacobsen et al., 2002a), and the apolipoprotein E codon 112 and 158 SNPs (Jacobsen et al., 2002b) as well as multiplex genotyping of 20 SNPs implicated in dysmetabolic syndrome and with Maturity Onset Diabetes of the Young (MODY) (Mouritzen et al., 2003). In the latter report, 40 allele-specific 12-mer LNA-substituted probes were designed with an LNA substitution located at the SNP position. Additional LNA nucleotides were used to increase and equalize the duplex melting temperature (Tm) across the SNP interrogation probes as well as to improve mismatch discrimination, while avoiding LNA in self-complementary sequences of the probes. In addition, LNA-substituted oligonucleotides have been used to improve the sensitivity and specificity in gene expression profiling by spotted 50-mer oligonucleotide microarrays (Tolstrup et al., 2003) and in gene knock down by LNA antisense (Wahlestedt et al., 2000; Braasch et al., 2002) and more recently, in efficient isolation of intact poly(A)+ RNA from lysed cell and tissue extracts by LNA oligo(T) affinity capture (Jacobsen et al., 2004).

The development of molecular probes and image analysis has made fluorescence in situ hybridization (FISH) a powerful investigative tool. Although FISH has proved to be a very useful technique in many areas, it is still a fairly time-consuming procedure with limitations in sensitivity and resolution. Thus, probes with higher affinity would potentially improve the sensitivity of the technique as reported for peptide nucleic acid (PNA) FISH probes (Taneja et al., 2001; Williams et al., 2002). We have previously shown that LNA-modified oligonucleotides are highly efficient as FISH probes (Silahtaroglu et al., 2003). Here we describe the LNA-FISH method in detail and report on the utility of LNA-modified FISH probes developed for different human repetitive elements, such as the centromeric, telomeric, and heterochromatic repeats as well as a low copy number repetitive sequence.

Materials and methods Chromosome preparations Chromosome preparations were made by standard methods from peripheral lymphocyte cultures. Slides were used within 5 days after preparation or stored in 100 % ethanol at – 20 ° C until use (Henegariu et al., 2001b). Synthesis of LNA probes The biotin- and Cy3-labeled DNA and LNA-substituted oligonucleotide probes (Table 1) were synthesized in 0.2-Ìmol scale on an Expedite synthesizer using the phosphoramidite method. Commercially available DNA and LNA phosphoramidite monomers and reagents were used following the con-

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Table 1. The LNA-FISH probes used in the present study. LNA: uppercase letters; DNA: lowercase letters

Probe ID

Target element

Probe sequence 5´-3´

Reporter

EQ 7358 EQ 7359 EQ 7360 EQ 10509 EQ 10508 EQ 7361 EQ 7362 EQ 7363 EQ 10455 EQ 13843 EQ 13844 EQ 13845 EQ 15444 EQ 14302 EQ 14195 EQ 14196

satellite-B satellite-B satellite-B satellite-B satellite-B satellite-B satellite-B satellite-B satellite-B telomere telomere telomere all centromeres chr13/21 BAC-1.LNA BAC-2.LNA

aTtccatTcgaTtccAttcgaTc aTtcCatTcgAtTccAttCgaTc aTTCcattcgATTccattcGATc aTtCcAtTcaGaTtCcAtTcGaTc attccattcgattccattcgatc aTtccatTcgaTtccAttcgaTc aTtcCatTcgAtTccAttCgaTc aTTCcattcgATTccattcGATc aTtCcAtTcGaTtCcAtTcGaTc ttagggttagggttagggttaggg TtAgGgTtAgGgTtAgGgTtAgGg tTaGgGtTaGgGtTaGgGtTaGgG ATGmCAGGTGGATA TgTgTaCcCaGcCaAaGgAgTtGa tCcGtGcCgTgTcCcTcGgCtCtCtcCcAtTc cAgAaCtTcTcCaAgGaTcAgCtCcCaAaTcAaC

5’-Biotin 5’-Biotin 5’-Biotin 5’-Biotin 5’-Cy3 5’-Cy3 5’-Cy3 5’-Cy3 5’-Cy3 5’-Cy3 5’-Cy3 5’-Cy3 5’-Biotin 5’-Biotin 5’-Biotin 5’-Biotin

ventional protocols (for details, see www.exiqon.com). After oligomerization, the biotin and Cy3 reporter groups were conjugated to the 5) end of the solid support-bound oligonucleotides using corresponding phosphoramidite and standard protocols recommended by the manufacturer. The oligonucleotides were subsequently removed from the solid support and deprotected by treatment with concentrated NH4OH and purified by RP-HPLC. Pretreatment of the slides Slides were either treated with RNAse A at 37 ° C for 2 h (Silahtaroglu et al., 1998) or with proteinase K for 10 min or pepsin (0.005 % in 0.01N HCl) for 1 min (Henegariu et al., 2001b) or in various combinations of the abovementioned treatments. Fluorescence in situ hybridization FISH was performed as described earlier with some modifications (Silahtaroglu et al., 2003). Ten microliters of biotin-labeled LNA-FISH probe was used at a concentration of 0.1–1 ÌM under a 20 × 20 coverslip. A simultaneous denaturation of the target DNA and the probe was carried out at 79 ° C for 2 min under the coverslip in the presence of the hybridization mixture containing 50 % formamide. Slides were hybridized for 30 min at 37 ° C. A hybridization oven (HYBrite Denaturation/Hybridization System; Vysis Inc, IL, USA) was used for denaturation and hybridization of the probe and the target. Post-hybridization washes were carried out at 65 ° C in 0.1× SSC for 3 × 5 min followed by a 5-min wash in 4× SSC/0.05 % Tween 20 at 37 ° C. The slides were incubated for 7 min with 1 % blocking reagent (Roche A/S, Hvidovre, Denmark), followed by incubation with fluorescein-conjugated avidin (Vector Laboratories, USA) for 10 min at 37 ° C. After a 3 × 3-min wash in 4× SSC/0.05 % Tween 20 at 37 ° C, and a 5-min wash in PBS, the slides were briefly dipped in water, dehydrated, air-dried and mounted in Vectashield (Vector Laboratories, USA) containing 4),6-diamidino-2-phenylindole (DAPI). The whole procedure was carried out in the dark. The signals were visualized using a Leica DMRB epifluorescence microscope equipped with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ), and IPLAB Spectrum Quips FISH software (Applied Imaging international Ltd., Newcastle, UK).

Results Human satellite-2 repeat The utility of LNA probes in FISH was initially evaluated using the human classical satellite-2 repeats on interphase nuclei and metaphase chromosomes (Silahtaroglu et al., 2003). Satellite-2 DNA, which is composed of multiple repeats of 23and 26-nt sequences, is especially concentrated in the large heterochromatic regions on chromosomes 1, 16, 9 and Y (Jean-

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Cytogenet Genome Res 107:32–37 (2004)

pierre, 1994). For the 23-nt human classical satellite-2 sequence (5)-attccattcgattccattcgatc-3)), five different oligonucleotide probes with different LNA substitution patterns were designed and synthesized containing either a 5)-Cy3 or 5)-biotin reporter (Table 1). Among the different LNA-modified oligonucleotides, the LNA-2 satellite-2 probe, in which every second nucleotide position was substituted with an LNA nucleotide (Table 1), clearly outperformed the other probes in sensitivity and consistently gave the best FISH results (Fig. 2a; see Silahtaroglu et al., 2003). Based on these results, we decided to use the LNA-2 substitution pattern for the remaining LNA-FISH probes used in this study (Table 1). Telomere FISH Telomeres are extraordinarily dynamic chromosomal structures.They are essential for genome stability and faithful chromosome replication and mediate many biological activities, including cell cycle regulation, cellular aging, movements and localization of chromosomes within the nucleus, as well as transcriptional regulation of subtelomeric genes. A conserved, (ttaggg)n tract forms the DNA component of each chromosome terminus in humans (Moyzis et al., 1988; Blasco et al., 1999). In the present study we designed two different LNA-2-substituted telomere probes, composed of four copies of the (ttaggg) repeat (Table 1), in which the LNAs were substituted at every second nucleotide (nt) position in phases 0 and 1, respectively. The phase 0 substituted LNA-FISH probe (5)-TtAgGgTtAgGgTtAgGgTtAgGg-3)) was shown to be much more efficient as a FISH probe than the probe substituted in phase 1 (5)-tTaGgGtTaGgGtTaGgGtTaGgG-3)), possibly due to the higher number of LNA-G substitutions and higher Tm in the latter resulting in unspecific binding to non-target sequences. Previously, improved telomere FISH has been reported using peptide nucleic acid (PNA) probes (Hultdin et al., 1998). We thus decided to compare the efficiency of LNA telomere FISH with a commercially available Telomere PNA FISH probe (DakoCytomation, Denmark). Direct comparison of the probes with LNA and PNA for telomere FISH demonstrated that the sensitivity and performance of the two probe types was fully comparable (Fig. 2b, c).

Fig. 2. FISH results using (a) human satellite-2 specific LNA-2 probe EQ 10455 gives signals on heterochromatic regions primarily on chromosomes 1, 16, 9, and weakly on Y and 15. (b) The 24-nt-long telomere-specific LNA-2 probe EQ 13844 and (c) telomere-specific PNA probe give signals on all telomeres (d). The human alpha-satellite-specific LNA probe EQ 15444 gives signals on all centromeres. (e) The chromosome 13/21-specific LNA-2 probe EQ 14302 stains the centromeres of chromosomes 13 and 21. (f) The LNA-2 probe EQ 14195 designed to detect a 32-nt-long repetitive sequence (copy number 37) gives signal on a chromosome 4q35.

Centromere FISH Centromeric DNA is often used for studying karyotypic evolution in various species, understanding the etiology of centric fusion chromosomes, as well as for delineating the origin and evolution of satellite DNA families and discriminating homologous chromosomes. Human centromeres are very

heterogeneous with respect to their nucleotide composition. The bulk of human centromeric DNA comprises tandemly arranged repetitive, satellite DNA and interspersed repetitive DNAs (Lee et al., 1997). The centromeres share a big bulk of DNA in common, yet many of the centromeres contain chromosome-specific repetitive sequences which are often uti-

Cytogenet Genome Res 107:32–37 (2004)

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lized in molecular cytogenetics (Willard, 1985; Kolvraa et al., 1991). For the present study we designed a fully substituted 11-nt LNA probe for the centromeric alpha-satellite sequence 5)ATGCAGGTGGATA-3) with a 5)-biotin reporter group. The LNA probe resulted in signals on all centromeres, whereas a second LNA alpha-satellite probe (5)-TgTgTaCcCaGcCaAaGgAgTtGa-3)) substituted with the LNA-2 design, specifically painted the centromeres of chromosomes 13 and 21 (Fig. 2d, e). FISH of tandem repeats In order to assess the resolving power of LNA-FISH, we made use of a repetitive sequence within an isolated BAC clone. The clone RP11-467J11, located at 4q35, was analyzed for repeat sequences (Tandem repeat finder; Benson, 1999), and subsequently LNA-2 substituted probes were synthesized for two of the repeats, which were 32 and 34 nt long with a copy number of 37 and 67, respectively (Table 1). Both LNA probes, separately or in combination, showed specific signals on chromosome 4q, when used in FISH (Fig. 2f), which indicates that the resolution of the LNA-enhanced FISH is at least in the order of 1–3 kb.

Discussion The expanding inventory of nearly 200 sequenced genomes to date have been the primary drivers in the recent explosion in genomics, resulting in the deconstruction of living organisms into comprehensive molecular catalogs of genes, transcripts and proteins. The importance of the genetic similarities between different species and the variation within a single species have become apparent with the completion of several important genome sequences, culminating in the publication of the working draft of the human genome in 2001 (International

Human Genome Sequencing Consortium, 2001; Sachidanandam et al., 2001; Venter et al., 2001). The human genome is much more complex than originally anticipated, comprising a wide variety of sequence features, such as regions of high-GC and of low-GC content, unique and repetitive sequences, protein-coding sequences, coding sequences for non-coding regulatory RNAs and non-coding functional elements. Around 50 % of the genome consists of repetitive sequences, such as transposon-derived interspersed repeats often referred to as interspersed repeats; processed pseudogenes, simple sequence repeats such as (A)n, (CA)n or (CGG)n, segmental duplications, consisting of 10–300-kb blocks of DNA that have been copied from one region of the genome into another region; and blocks of tandemly repeated sequences, such as those at centromeres, telomeres, the short arms of the acrocentric chromosomes and ribosomal gene clusters (International Human Genome Sequencing Consortium, 2001; Science Genome Map, 2001). FISH has proved to be an efficient technique for studying repetitive sequences in metaphase or interphase nuclei (Silahtaroglu et al., 1998; Henegariu, 2001a). With the aim of improving the sensitivity and resolution of FISH, we have designed and developed LNA-modified FISH probes for the investigation of different tandem repetitive sequence elements, such as the satellite-2 repeats in heterochromatic regions, the alpha-satellite repeats in centromeres and the telomeric repeats. We report here that the substitution of short DNA oligonucleotides with the high-affinity RNA analog LNA results in highly efficient and sensitive FISH probes with a wide range of applications in detecting various repetitive elements in the human genome. The results obtained with tandem repeats indicate that the resolution of LNA-enhanced FISH is at least in the order of 1–3 kb. Given its high affinity, efficiency and sensitivity, LNA-FISH could potentially be used in the detection of single nucleotide polymorphisms, mutations or size polymorphisms at the chromosome level.

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