Design of LNA probes that improve mismatch discrimination

You, Yong; Moreira, Bernardo G.; Behlke, Mark A.; Owczarzy, Richard

doi:10.1093/nar/gkl175

Cited by 285 publications

(316 citation statements)

References 35 publications

(72 reference statements)

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“…We used different types of nucleic acids targeting H. pylori: LNA, 2′OMe, unlocked nucleic acid (UNA), PNA, and DNA (Table 1). Different designs of oligo-nucleotide probes were tested to find the best discrimination possible based on earlier published criteria to improve mismatch discrimination You et al 2006;Langkjaer et al 2009;Wengel 2010, 2011;Owczarzy et al 2011). As DNA has a restricted flexibility of oligonucleotide design, only one probe was designed (Table 1).…”

Section: Probe Designmentioning

confidence: 99%

“…In this case, and because there are no guidelines related to the minimum ΔG°, we based our design on our extensive experience in working with these probes (Guimaraes et al 2007;Cerqueira et al 2011Cerqueira et al , 2013. In contrast, LNA, 2′OMe, and UNA bases can be positioned anywhere within an oligonucleotide sequence, which means that the design is much more flexible and that probe fine-tuning is possible (You et al 2006). As a general rule, these probes were designed with a triplet of LNA-modified nucleotides positioned at the center of the mismatch site to improve the discrimination (You et al 2006;Owczarzy et al 2011).…”

Section: Probe Designmentioning

confidence: 99%

“…In contrast, LNA, 2′OMe, and UNA bases can be positioned anywhere within an oligonucleotide sequence, which means that the design is much more flexible and that probe fine-tuning is possible (You et al 2006). As a general rule, these probes were designed with a triplet of LNA-modified nucleotides positioned at the center of the mismatch site to improve the discrimination (You et al 2006;Owczarzy et al 2011). The use of a higher density of LNA residues in each probe is an-other important parameter to improve mismatch discrimination (You et al 2006).…”

Section: Probe Designmentioning

confidence: 99%

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Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

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Section: Probe Designmentioning

confidence: 99%

Section: Probe Designmentioning

confidence: 99%

Section: Probe Designmentioning

confidence: 99%

See 1 more Smart Citation

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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“…33 Once proven the pathogenic effect of the c.820 þ 13A4G mutation, we investigated the possibility of restoring correct splicing by antisense therapy. LNA oligonucleotides were chosen for their highmismatch discrimination ability 34 and their high stability and low toxicity in biologic systems. 35 This variation activates a cryptic donor splice site extremely close to the natural one, thus its steric blocking by antisense oligonucleotides could affect the correct splicing.…”

Section: Discussionmentioning

confidence: 99%

Functional correction by antisense therapy of a splicing mutation in the GALT gene

et al. 2014

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In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820 þ 13A4G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. European Journal of Human Genetics (2015) 23, 500-506; doi:10.1038/ejhg.2014.149; published online 23 July 2014 INTRODUCTIONOver the last years, splicing mutations emerged as an important pathogenic mechanism, underlying 10-30% of genetic diseases (HGMD Professional 2013.1). 1 Splicing accuracy depends not only on the recognition of exon-intron junctions, defined by intronic ciselements: 5 0 splice site, 3 0 splice site, branch site and poly-pyrimidine tract, 2-4 but also on more discrete elements, entitled splicing regulatory elements, which direct the splicing machinery to use the correct splice sites. Exonic and intronic splicing enhancers stimulate splicing and serve as binding sites mainly for serine/arginine-rich proteins. Exonic and intronic splicing silencers repress splicing, and often function by binding proteins from the heterogenous nuclear ribonucleoprotein family. 2,4-6 Although most reported splicing mutations directly abolish an authentic splice site or create a novel one, an increasing number of disease-associated variations that alter splicing enhancers or silencers have been reported. 2,7-9 Each nucleotide modification should be considered a potential candidate for splicing alterations, as not only intronic but also nonsense, missense and silent modifications may impact splicing. 7 Accordingly, constitutive and regulated splicing reactions are considered potential therapeutic targets and novel strategies for their correction are evolving. Among these, antisense oligonucleotides display an exquisi...

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“…17 The ⌬T m s between matched and mismatched PNA/DNA or LNA/DNA duplexes usually exceed 10°C, sufficient for the selection of minor variant alleles with sensitive detection. 16,18 Unlike other commonplace methods, such as amplification refractory mutation system PCR, this method can enrich different base changes on the target sites using a single wildtype-specific PNA/LNA. In principle, this does not aberrantly alter the wild-type sequences and leads to much lower false-positive rates.…”

Section: Discussionmentioning

confidence: 99%

Mutant Enrichment with 3′-Modified Oligonucleotides

Lee

Kim

Kown

et al. 2011

The Journal of Molecular Diagnostics

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Many clinical situations necessitate highly sensitive and reliable molecular assays; however, the achievement of such assays remains a challenge due to the inherent limitations of molecular testing methods. Here, we describe a simple and inexpensive enrichment technique that we call mutant enrichment with 3=-modified oligonucleotides (MEMO). The method is based on the use of a 3=-modified oligonucleotide primer that blocks extension of the normal allele but enables extension of the mutated allele. The performance of the technique was evaluated with respect to its ability to detect common cancer mutations in the EGFR, KRAS, BRAF, TP53, JAK2, and NPM1 genes. We achieved sensitivities of 10 ؊2 to 10 ؊6 using downstream Sanger sequencing, depending on the concentrations and thermodynamics of the primers. MEMO

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Design of LNA probes that improve mismatch discrimination

Cited by 285 publications

References 35 publications

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Functional correction by antisense therapy of a splicing mutation in the GALT gene

Mutant Enrichment with 3′-Modified Oligonucleotides

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