Introduction:: Rapid, accurate detection of disease-related nucleic acids (NA) is critical in clinical diagnostics. A variety of NA biosensors have been designed based on the hybridization of complementary NAs and the activity of different enzymes, including the standard Polymerase Chain Reaction (PCR)1 as well as isothermal amplification reactions like Loop Mediated Amplification Reaction (LAMP)2-3, Exponential Amplification Reaction (EXPAR)4, and Ultrasensitive DNA Amplification Reaction (UDAR)5-6. For RNA detection, the standard method is to first transduce the RNA into complementary DNA by reverse transcriptase. However, quantification inaccuracies can occur with low abundant targets and between highly conserved sequences7. This is a challenge for an emerging class of biomarkers known as microRNA (miRNA), which are small (~22 nucleotides) and have highly conserved sequences within families, requiring a sensitive and specific assay for accurate detection8. In this work, we explored a new transduction method where the RNA target hybridizes with two complementary DNA templates to facilitate ligation of a new, long transduction template (Fig 1A). We tested this method to detect miRNA in combination with Ultrasensitive DNA Amplification Reaction (UDAR), a nonlinear, biphasic reaction developed in our lab. We hypothesized that this strategy would increase specificity for the target miRNA through the three-strand complex and increase miRNA transduction efficiency by using a DNA primer rather than extending from the miRNA itself, which has less affinity for the polymerase. This versatile transduction method is also broadly applicable for many types of single stranded RNA targets in a variety of amplification reactions.
Materials and Methods:: For all experiments, target miRNA was diluted in yeast RNA and mixed with TT1, TT2, and primer DNA (Fig 1A) then annealed by incubating at 95oC for 5 minutes and cooling to 25oC at 0.1oC/second. UDAR reaction buffer contained 1x ThermoPol® I Buffer, 25 mM Tris-HCl (pH 8), 6 mM MgSO4, 40 mM KCl, 0.1 mg*mL-1 BSA, 6.33% glycerol, 100 mM trehalose, and 7.5 mM TMAC. Ligation was tested in either 1x SplintR Ligase Reaction buffer (NEB) or 1x UDAR buffer for either 30 or 60 minutes at 25oC or 37oC, using denaturing PAGE to quantify relative ligation efficiency at the different conditions. Final reaction mix contained 10pM miRNA, 10nM each TT1, TT2, and primer, 1x UDAR buffer, 0.5 mM each dNTPs, 2x Evagreen dye, 0.2 U*µL-1 Nt·BstNBI, 0.0267 U*µL-1 Bst 2.0 WarmStart® DNA Polymerase, SplintR Ligase at various concentrations, and 50nM exponential UDAR template in a final volume of 20 uL. Samples were ligated at 25oC for 30 minutes, then incubated at 55oC for 80 minutes to create a large nonlinear signal using UDAR. Fluorescence measurements were taken every 32 seconds. Fluorescent traces were analyzed using custom Python code to quantify inflection points and separation time between positive and negative samples. Primer and TT2 designs were varied for primer lengths of 10, 12, 15, and 20 nucleotides with 0, 2, and 4 nucleotide separation between the primer and miRNA binding sites. Each reaction was done in triplicate with negative controls containing either no ligase or non-specific miRNA let7f.
Results, Conclusions, and Discussions:: We designed a novel miRNA transduction method with two DNA transduction templates, each complementary to half of the target miRNA (Fig 1A). The first template TT1 contains a primer binding site. The second template TT2 contains the complementary sequence for a nickase recognition site. The miRNA-TT1-TT2 complex allows SplintR ligase to form the TT1/2 strand. A polymerase can extend from a primer along TT1/2 to create the recognition site. A nicking enzyme cleaves at this site to release a short DNA product which can initiate UDAR, a nonlinear DNA amplification reaction. We confirmed ligation at various times, temperatures, and buffer conditions using PAGE (Fig 1B). The ligation efficiency was not significantly different between conditions and there was no significant presence of unwanted ligation products, however the conversion of TT1 and TT2 to TT1/2 was low. Next, we compared 100nM, 175nM, and 250nM ligase in the full amplification reaction. The lower concentrations had increased separation time between positive and negative samples compared to 250nM, possible due to lower concentrations of ligase buffer components which might inhibit amplification (Fig 1C). Finally, target miR223 was detected with the full transduction-UDAR reaction for four different primer/TT2 designs. The presence of ligase appears to slow the reaction compared to samples without ligase, except for the 15nt primer design (Fig 1D). This indicates that ligation is not required for amplification and that miRNA is still priming the reaction, and again that ligase buffer components may inhibit amplification. Additional optimization is needed to increase ligation efficiency, providing higher TT1/2 concentrations to better catalyze the reaction. Amplification in the presence and absence of each oligonucleotide and with various buffer additives would help identify how to create a ligation-dependent reaction. In conclusion, this miRNA detection method aims to increase the specificity and sensitivity of detection assays by requiring a three-strand complex to initiate ligation and amplification. Initial results highlight critical experimental parameters of assays using SplintR ligase and reaffirm the importance of including controls without ligase when optimizing ligation-based detection assays. Our finalized transduction method will be widely applicable for single-stranded NA detection in a variety of amplification reactions.
Acknowledgements (Optional): : Supported by the National Science Foundation under Grant No. 1847245 and the Montana State University Kopriva Graduate Fellowship
References (Optional): : 1. Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., & Hellens, R. P. (2007). Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant methods, 3(1), 1-12.
2. Notomi, T., Mori, Y., Tomita, N., & Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of microbiology, 53(1), 1-5.
3. Huang, W. E., Lim, B., Hsu, C. C., Xiong, D., Wu, W., Yu, Y., ... & Cui, Z. (2020). RT‐LAMP for rapid diagnosis of coronavirus SARS‐CoV‐2. Microbial biotechnology, 13(4), 950-961.
4. Van Ness, J., Van Ness, L. K., & Galas, D. J. (2003). Isothermal reactions for the amplification of oligonucleotides. Proceedings of the National Academy of Sciences, 100(8), 4504-4509.
5. Özay, B., Robertus, C. M., Negri, J. L., & McCalla, S. E. (2018). First characterization of a biphasic, switch-like DNA amplification. Analyst, 143(8), 1820-1828.
6. Özay, B., Murphy, S. D., Stopps, E. E., Gedeon, T., & McCalla, S. E. (2022). Positive feedback drives a secondary nonlinear product burst during a biphasic DNA amplification reaction. Analyst, 147(20), 4450-4461.
7. Lee, I., Baxter, D., Lee, M. Y., Scherler, K., & Wang, K. (2017). The importance of standardization on analyzing circulating RNA. Molecular diagnosis & therapy, 21, 259-268. 8. Qin, X., Wang, X., Xu, K., Zhang, Y., Tian, H., Li, Y., ... & Yang, X. (2023). Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR. Molecular Therapy-Nucleic Acids, 31, 241-255.