LNA Nucleotides Research Articles

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2'-fluoro and Locked Nucleic Acid Nucleotides.

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B.

SummarySensitive and accurate RT‐qPCR tests are the primary diagnostic tools to identify SARS‐CoV‐2‐infected patients. While many SARS‐CoV‐2 RT‐qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands‐on‐time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS‐CoV‐2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS‐CoV‐2 RT‐qPCR tests that are originally based on the protocol targeting regions of the RNA‐dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS‐CoV‐2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature‐stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction‐free version enables high sensitivity detection of SARS‐CoV‐2 in about an hour using minimally invasive, self‐collected gargle samples. These RT‐qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS‐CoV‐2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Towards the enzymatic synthesis of phosphorothioate containing LNA oligonucleotides

Therapeutic oligonucleotides require the addition of multiple chemical modifications to the nucleosidic scaffold in order to improve their drug delivery efficiency, cell penetration capacity, biological stability, and pharmacokinetic properties. This chemical modification pattern is often accompanied by a synthetic burden and by limitations in sequence length. Here, we have synthesized a nucleoside triphosphate analog bearing two simultaneous modifications at the level of the sugar (LNA) and the backbone (thiophosphate) and have tested its compatibility with enzymatic DNA synthesis which could abrogate some of these synthetic limitations. While this novel analog is not as well tolerated by polymerases compared to the corresponding α-thio-dTTP or LNA-TTP, α -thio-LNA-TTP can readily be used for enzymatic synthesis on universal templates for the introduction of phosphorothioated LNA nucleotides.

Comparison of different chemically modified inhibitors of miR-199b in vivo

MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3′ to the 5′ end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5′ end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.

Determination of variations of the primary structure of the UGT1A1, DPYD, GSTP1, and ABCB1 genes involved in the metabolism of antitumor agents

A individual approach to the treatment of cancer patients, which takes the genetic features of the patient’s metabolism into account, allows one to choose an appropriate drug and its dose, that increases the efficiency of therapy and prevents the development of side toxic effects. For this purpose, we developed a biological microarray-based method for studying the primary structure of patient DNA and identifying polymorphisms in the UGT1A1, DPYD, GSTP1, and ABCB1 genes associated with alterations in xenobiotic metabolism. Genotyping of samples from 89 cancer patients and 15 healthy donors was carried out using the developed microarray. The results of determination of the primary structure of DNA samples coincided completely with the control sequencing. To increase the specificity of determination of polymorphic variants of the UGT1A1 gene, we used hybridization probes containing LNA nucleotides. The frequencies of polymorphic allelic variants of the UGT1A1*28, DPYD*2A, GSTP1 (I105V), and ABCB1 (C3435T) genes in the studied sample were 0.39, 0, 0.33, and 0.57, respectively.

Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2'-O-Methyl RNA and RNA Oligonucleotides.

This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes.

Selection of LNA-containing DNA aptamers against recombinant human CD73.

LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

Improvement of a streptavidin-binding aptamer by LNA- and α-l-LNA-substitutions

Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and compared with the unmodified DNA aptamers. A ∼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3′-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability.

LNA-based in situ hybridization detection of mRNAs in embryos.

In situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.

Crystallographic characterization of CCG repeats

CCG repeats are highly over-represented in exons of the human genome. Usually they are located in the 5′ UTR but are also abundant in translated sequences. The CCG repeats are associated with three tri-nucleotide repeat disorders: Huntington’s disease, myotonic dystrophy type 1 and chromosome X-linked mental retardation (FRAXE). In this study, we present two crystal structures containing double-stranded CCG repeats: one of an RNA in the native form, and one containing LNA nucleotides. Both duplexes form A-helices but with strands slipped in the 5′ (native structure) or the 3′ direction (LNA-containing structure). As a result, one of two expected C-C pairs is eliminated from the duplex. Each of the three observed C-C pairs interacts differently, forming either one weak H-bond or none. LNA nucleotides have no apparent effect on the helical parameters but the base stacking is increased compared to the native duplex and the distribution of electrostatic potential in the major groove is changed. The CCG crystal structures explain the thermodynamic fragility of CCG runs and throw light on the observation that the MBNL1 protein recognises CCG runs, as well as CUG and CAG, but not the relatively stable CGG repeats.

Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand–duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form.

Expression Analysis of WT1 Splicing Variants In CML and AML: −5/+KTS as a Novel Candidate for Early Marker of Relapse

AbstractAbstract 2749Total WT1 expression is currently used for monitoring of AML patients and is of prognostic value also for CML patients according to our experience. Total WT1 level brings together levels of enormous amount of WT1 variants. Four major splicing variants reffered to as −5/−KTS, −5/+KTS, +5/−KTS and +5/+KTS are produced by combining two main independent splicing sites – exon 5 and KTS sequence. Short WT1 (sWT1) variant originates from usage of an alternative first exon E1a (Dalloso et al., 2001). Interestingly, exon 5 splicing was associated with resistance to apoptosis (Pritchard et al., 2006). The sWT1 was reported as overexpressed specifically in leukemias (Hossain et al., 2006). In view of literature data, we have suggested that splicing variant ratio or particular variant levels might have impact to leukemia pathogenesis, influence responsiveness to therapy and thus be a candidate for novel leukemic markers giving additional or more precise information about the disease state besides total WT1 levels. This idea was enhanced by our previous observation of differences of the four major variants levels associated with risk groups and different achieved responses to therapy among diagnostic AML patients samples (ASH 2009). In the current study, we extended our field of interest also on sWT1 vs. full length WT1. We aimed to find out whether there might be any advantage of monitoring of any of the WT1 variant in AML and CML patients undergoing therapy for more precise, sure and optionally earlier relapse detection/prediction.We have designed a novel method for quantification of sWT1 and full length WT1 separately. Quantification is performed by two separated real-time PCRs based on discriminating forward primers. One of those primers hybridizes onto exon 1 the other onto alternative exon 1a which enables to quantify full length WT1 and sWT1, respectively. For quantification of the four major variants, we used a method that we previously developed and reported (ASH, 2009). By introducing LNA nucleotides into the sequence of the primers with lowest hybridization efficiency we further improved reaction sensitivity, efficiency and robustness. Fourteen CML and ten AML patients undergoing therapy and developing haematological or molecular relapse were retrospectively analysed so far.We found out that the ratio of the four variants was changed in relapse as compared to diagnosis. The most abundant variant at diagnosis was +5/−KTS. In relapse, it became to be −5/+KTS in both AML and CML. The −5/+KTS was the first one to be increased in most of the cases and usually its expression kinetics was the most expressive of all four variants. As compared to BCR-ABL transcript, both total WT1 and −5/+KTS increases were more expressive or even earlier in some of CML patients and thus clearly predicted upcoming relapse. Short WT1 was detected in all AML samples, in CML we found it only in blast crisis not in the chronic phase samples. In all cases the proportion of sWT1 from total WT1 was very low, the majority of total WT1 was represented by full length WT1.Taken together, we showed that mainly −5/+KTS WT1 variant is a good marker of CML and AML and well characterizes the course of the diseases. Our preliminary data indicated that both kinetics and actual levels of −5/+KTS might specify information done by total WT1 levels and in case of CML by BCR-ABL making relapse prediction more sure or possibly even earlier.Supported by MZOUHKT2005. Disclosures:No relevant conflicts of interest to declare.

Polymerase directed incorporation studies of LNA-G nucleoside 5′-triphosphate and primer extension involving all four LNA nucleotides

For the first time, the enzymatic incorporation of LNA-G nucleotides into DNA strands has been investigated. KOD DNA polymerase accepted LNA-GTP as a substrate and provided a full-length dsDNA product modified with LNA-G nucleotides. Most importantly, an extension experiment was conducted using a reaction mixture containing LNA-A, LNA-T, LNA-G and LNA-mC nucleotides as the only NTP sources and this showed that KOD DNA polymerase can even incorporate 21 LNA nucleotides successively to a full-length extension product. Also, the experiments revealed that LNA-containing extension products were much more resistant towards the 3′→5′ exonucleolytic cleavage activity of the enzyme than DNA extension products.

Efficient enzymatic synthesis of LNA-modified DNA duplexes using KOD DNA polymerase

Three different LNA-nucleoside triphosphates, LNA-TTP, LNA-ATP and LNA-5-methyl-CTP, were investigated as substrates for KOD DNA polymerase. The results reveal that KOD DNA polymerase is an efficient catalyst for template directed synthesis of DNA oligonucleotide duplexes containing a large number of LNA nucleotides by primer extension reactions. Furthermore, KOD DNA polymerase is shown to be suitable for the PCR amplification of an LNA-modified DNA duplex.

Enzymatic Incorporation of LNA Nucleotides into DNA Strands

Unlocking uses of locked nucleic acids: LNA nucleoside 5′-triphosphates have been synthesized, and their ability to serve as substrates for polymerases have been investigated. Phusion high-fidelity DNA polymerase was found to be an efficient enzyme for incorporating LNA nucleoside 5′-triphosphates into DNA strands.

Rapid and easy prenatal diagnosis of sickle cell anemia using double‐dye LNA probe technology

Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in sub-Saharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Specificity of the probes used in this approach. 20 ng of genomic DNA from three patients homozygous for the wild-type (WT/WT), S (S/S) and C (C/C) alleles respectively was tested in this experiment. The results given by the each probe are illustrated on different panels. The Cy5 channel was applied for the analysis of the WT probe, while FAM and Yakima Yellow were used for the S and C probes, respectively. The three probes showed absolute specificity because there was no signal detected when amplified genomic DNA did not contain the nucleotide targeted by the probe. The no template control did not give any signal for the three probes either. Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instruments that are widely used for multicolour real-time PCR analysis. The results obtained on the LC480 (Roche, Basel, Switzerland), the iCycler (Bio-Rad, Hercules, CA, USA) and the M × 3000 p (Stratagene, La Jolla, CA, USA) were perfectly comparable from one machine to another (see Fig S1). Although a fast PCR protocol was not applicable on all these machines, a longer PCR protocol did not affect the results. In summary, we have developed a very robust and rapid method that takes advantage of the high specificity of double-dye LNA probes, which enabled the identification of the two main mutations in SCD in <40 min. The protocol developed in our laboratory is easily transferable to other real-time PCR machines. The high sensitivity of our approach represents a great advantage for the analysis of samples containing small amounts of DNA. Moreover, this single closed tube method offers another obvious advantage for prenatal diagnosis as it prevents risk of cross contamination. We gratefully acknowledge the skillful technical assistance of Sandrine Delbauve. Table SI. Oligonucleotides used for the three-colour real-time polymerase chain reaction (PCR) assay in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Mutation JAK2 V617F in Patients with Myeloproliferative Disorders and Technical Aspects of Its Detection.

Myeloproliferative disorders (MPD) are a heterogeneous group of diseases that involve chronic myeloid leukemia (CML), polycythemia vera (PV), essential trombocythemia (ET), chronic idiopatic myelofibrosis (IMF), hypereosinophilic syndrome and chronic neutrophilic leukemia. Recently, it has been found that in Ph-negative MPD, acquired somatic mutation in JAK2 gene can be identified, causing substitution of valine for phenylalanine at aminoacid position 617 (V617F). Experimental evidence indicates that JAK2 V617F might cause erythropoietin-independence of erythroid colonies and receptor hypersensitivity, thus be implicated in the pathogenesis of PV.The incidence of V617 in PV is high, reaching 96% of cases; in ET or IMF, the mutation is less frequent. Nevertheless, the percentage of JAK2 V617F-positive patients differs greatly among studies, depending on the detection technique used. Sequencing analysis, as one of the options, can underrate the number of positive cases, as its sensitivity in authentic samples with varying proportion of mutant granulocytes is unsatisfactory. The nucleotide context of the V617F site is complicated (direct repeat TGTG/TT), and thus prone to sequencing artifacts.For specific and sensitive detection of V617F mutation in JAK2 gene, we have developed a novel allelic discrimination assay, based on the usage of Real-Time PCR technology and LNA-modified fluorescently labeled probes (Locked Nucleic Acids). This technology is distinguished by 100% discrimination efficiency, and sensitivity detection rate reaching 10% of mutant granulocytes in an authentic sample. LNA-modified probes are extremely powerful tool for precise genotyping, since they are short (12-mers), and due to the interspersed LNA nucleotides, melting temperature (Tm) of the probe/target hybrid is high. Hence, the LNA-based genotyping technology combines the desired features of high sensitivity and absolute specificity.Herein, we present an analysis of JAK2 V617F detection in our group of 157 consecutive patients with the diagnosis of MPD or suspected MPD. Using our novel LNA-based Real-Time PCR genotyping technique, we were able to identify varying proportions of the mutant JAK2 allele in 87 cases (55.4%); in the remaining 70 patients the mutation was not detected.The LNA-based Real-Time PCR technique is extremely precise and efficient in identifying even subtle proportion of JAK2 V617F mutant granulocytes, which in cases of diagnostic uncertainties on the origin of the polycythemia might be of great importance.

Solution structure of a dsDNA:LNA triplex

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5'-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19 degrees C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid.

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.