Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Abstract

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.

Folding Stability and Self‐Association of a Triplet‐Repeat (CAG)20 RNA Hairpin in Cytomimetic Media

“…These findings are in accordance with those of Furlan et al [26] . In their study, organic fluorophores were shown to significantly stabilize a small 10 bp DNA duplex.…”

“…These findings are in accordance with those of Furlan et al [26] In their study, organic fluorophores were shown to significantly stabilize a small 10 bp DNA duplex. However, these effects were found to decrease with increasing size of the nucleic acid construct, being a lot less decisive for a 21 bp duplex.…”

Circulating MicroRNAs as Promising Diagnostic Biomarkers for Patients With Glioma: A Meta-Analysis

“…Secondly, there was no consensus about the unified internal reference, leading to inconsistent results in miRNA relative quantitative analysis. Thirdly, the conventional and standard method of miRNA detection was the qRT-PCR method, characterized by complicated, tedious, and time-consuming manipulation (64), high experimental requirements, and the use of RNA which easily degrades, which limited extensive clinical practice (65). Hence, some novel methods need to be developed for implementing rapid miRNA detection with high sensitivity and selectivity, such as a toehold-mediated strand displacement reaction (SDR) (64), the enzyme-free surface plasmon resonance imaging (SPRi) biosensing method (66), and the ultrasensitive electrochemical method (67), etc.…”

Detection of unamplified target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor

“…In addition, quantitative analysis of these nucleic acid amplification techniques often requires sophisticated probe/primer design and optimization methodologies for efficient targeting. Furthermore, they often still rely on large and expensive optical components for detection 9,10 . Therefore, new methodologies are needed to overcome the limitations associated with conventional nucleic acid detection strategies to afford low-cost and fully integrated compact nucleic acid-based diagnostic tools to expand their clinical utility.…”