Biologically stable threose nucleic acid-based probes for real-time microRNA detection and imaging in living cells

MicroRNAs are tiny RNA molecules (miRNA) that influence post-transcriptional gene expression by silencing messenger RNA (mRNA). This novel class of molecules have an important role in a an array of biological processes, including cell proliferation, differentiation, and death and changes in their expression can contribute to disease development.  Indeed, miRNA is believed to be possess an array of advantages that could turn them into ideal candidates for biomarkers in a variety of afflictions. An ideal biomarker needs to be easily accessible, a condition that applies to miRNAs that can easily be extracted through liquid biopsies from blood, urine and other bodily fluids. It also has a high specificity for the tissue or cell type of provenance and it is sensitive in the way that it varies according to the disease progression,

The analytical methods for miRNA analysis include quantitative reverse transcription polymerase chain reaction (qRT-PCR), northern blotting, microarrays, and next-generation sequencing (NGS), all of which have drawbacks that restrict their practical applicability. The value of direct imaging of miRNA in living cells for detecting malignant cells and monitoring drug effectiveness in real time is still until recently not realized. As a result, a rapid, simple, cost-effective, and sensitive approach for in situ detection of miRNA expression is being developed by various investigators.

Enzymatic degradation, nuclear entrapment, a high false-positive signal, cytotoxicity, and the requirement for transfection agents are all issues that must be resolved before they may be used as diagnostic tools. To improve applicability, previous investigations have used Xeno nucleic acids (XNAs), chemically modified nucleic acid analogues such as locked nucleic acid (LNA), peptide nucleic acid, and O-methyl RNA as building blocks to construct biosensors that detect endogenous RNAs in live cells. In terms of thermal stability, binding affinity, and selectivity toward target RNAs, these chemically modified nucleic acid analog-based markers outperform DNA-based markers, resulting in shorter detection times and lower detection limits on the femtomolar scale. The study of RNA detection methods based on XNA is still in its early phases. QIAGEN’s miRCURY LNA miRNA Detection Probes are now the only commercially available miRNA detection probes (USA).

The design of LNA oligomers is hampered by several constraints: (1) sequences of more than four LNA nucleotides must be avoided; (2) sequences of three or more Cs or Gs must be avoided; (3) the GC content must be limited to 30% to 60%; and (4) self-complementarity or cross-hybridization must be avoided. The number of LNA oligomer sequences that can be synthesized and used as capture probes in biosensors is restricted, making it difficult to detect certain changes in genes of interest. As a result, commercially available LNA-based probes cannot be used as high-throughput biosensors in biotechnology or medicine.

In a new study published in Molecular Therapy by Fei Wang, Ling Sum Liu, Pan Li, Hoi Man Leung, Dick Yan Tam, and Pik Kwan Lo from the City University of Hong Kong, demonstrated the feasibility of employing threose nucleic acid (TNA) as a component in the construction of a biocompatible probe for fluorescence detection and imaging of intracellular RNA targets. TNA, a XNA-based biosensor, possess excellent physiological stability, high specificity and affinity towards RNA, robust enzymatic resistance, and effective cellular absorption. These TNA probes can be used not only detect and quantify dynamic expression of malignant cells miRNAs in cancer cell lines, but also to differentiate between cell lines, which is a key objective in the domain of live cell analysis. For constant real-time monitoring of target miRNAs, TNA probes could be efficiently taken up by living cells with minimal damage. Furthermore, the TNA probes were able to distinguish between different target miRNA expression levels in tumor cell lines.

The research team successfully created TNA-based probes for rapid and accurate target miRNA detection and imaging in living cells. This is also the first time that hybridization-based probes for target miRNA detection have been produced utilizing TNA’s base-pairing capabilities to natural nucleic acids. As TNAs have such great binding specificity and affinity for their matching RNA sequences, strand displacement would almost certainly occur when longer miRNA targets are bound to the partially hybridized TNA strand in TNA probes. As an output signal, this process would produce fluorescence enhancement. Researchers proposed that TNA-based probes due to their remarkable storage ability are essential for long term cellular studies. Investigators also claim the procedure to synthesize TNA to be cost efficient.

According to the authors, the new work by Professor Pik Kwan Lo and colleagues developed TNA-based probes for miRNA detection and imaging in live cells that are both robust and sensitive. This research also shows that TNA probes were employed for the first time to produce a physiologically stable and cost-effective biosensor for target miRNA detection and intracellular imaging as well as to enhance TNA molecule production and use in biological and biomedical research. It is hopefully that miRNAs probes such as the one reported in the new study will expedite the application of miRNA detection to become a routine approach in the development of personalized patient profiles, therefore allowing targeted therapeutic interventions.