“Clickable” LNA/DNA probes for fluorescence sensing of nucleic acids and autoimmune antibodies

Jørgensen, Anna Søndergaard; Gupta, Pankaj; Wengel, Jesper; Astakhova, Kira

doi:10.1039/c3cc45507f

Cited by 29 publications

(51 citation statements)

References 19 publications

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“…In light of the immense development of genomics and biomedicine, nucleic acid targeting represents a powerful diagnostic and therapeutic approach with high translational potential . In particular, the ability to design specific nucleic acid sequences to detect cancer‐ and virus‐associated genes quantitatively facilitates personalised medicine approaches with a high prognostic and diagnostic potential .…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, advances in gene therapy have increased the potential of nucleic acid targeting by synthetic DNA and RNA oligomers . Given these advances, the stage has been set to translate the information garnered from sequencing studies into personalised diagnostic and treatment strategies . In particular, human oncogenes have been extensively investigated, and many promising preclinical studies have shown clinical association of the mutations in these oncogenes with disease progression and treatment outcome .…”

Section: Introductionmentioning

confidence: 99%

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Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 μm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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“…Simultaneously the price of the probe increases fivefold or more, along with decreased photostability and chemical stability, since most quenchers of fluorescence are rather unstable molecules. We also demonstrated the improved sensitivity of the novel probes to SNPs compared to 5 0 -labeled analogues (Jørgensen et al 2013;Okholm et al 2014) (Fig. 8).…”

Section: Dna Groove-binding Dyesmentioning

confidence: 81%

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

Astakhova

2015

RNA Technologies

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“…In addition, as with the (phenylethynyl)pyrene probes described above, these probes have strong sensitivity to SNPs in the target RNAs. To harness this characteristic further, we synthesized "clickable" 2′-amino-LNA derivatives for direct attachment of fluorophores to the 2′-amino-position via a 1,2,3-triazole linker ( Figure 3D) [27]. As we demonstrate in a recent paper [27], attaching the fluorophores directly to the fluorescently labelled 2′-amino-LNA nucleotides improves the SNP discrimination of DNA and RNA targets.…”

Section: Enzyme-free Hybridization Assays: Specificity In Focusmentioning

confidence: 99%

“…To harness this characteristic further, we synthesized "clickable" 2′-amino-LNA derivatives for direct attachment of fluorophores to the 2′-amino-position via a 1,2,3-triazole linker ( Figure 3D) [27]. As we demonstrate in a recent paper [27], attaching the fluorophores directly to the fluorescently labelled 2′-amino-LNA nucleotides improves the SNP discrimination of DNA and RNA targets. This makes them a reliable tool for allele-specific detection of nucleic acids by fluorescence microscopy.…”

Section: Enzyme-free Hybridization Assays: Specificity In Focusmentioning

confidence: 99%

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

Miotke

Barducci²,

Astakhova³

2015

Chemosensors

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Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

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“Clickable” LNA/DNA probes for fluorescence sensing of nucleic acids and autoimmune antibodies

Abstract: Herein we describe fluorescent oligonucleotides prepared by click chemistry between novel alkyne-modified locked nucleic acid (LNA) strands and a series of fluorescent azides for homogeneous (all-in-solution) detection of nucleic acids and autoimmune antibodies.

Cited by 29 publications

References 19 publications

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

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