"HIGH-THROUGHPUT AND MULTIPLEX ANALYSIS OF SINGLE-CELL, SINGLE-MITOCHONDRIAL DNA MUTATION USING HYDROGEL DROPLET MICROFLUIDICS AND ROLLING CIRCLE AMPLIFICATION"

While understanding mitochondrial DNA (mtDNA) heteroplasmy and intercellular heterogeneity requires singlecell, single-mtDNA analysis, existing methods restrict studies to small cell populations [1,2].Water-in-oil droplet microfluidics improved the throughput of single cell analysis >100,000 cells, but it has been challenging for subcellular organelle analysis as their hierarchy in single-cell level should be resolved by multistep bioassays [3].Hydrogel droplet microfluidics has great potential for sub-cellular molecule analysis as they allow single-cell compartmentalization even after multiple times of buffer exchange steps.Here, we present a high-throughput, multiplex method for single-cell, single-mtDNA mutation analysis.(Figure 1).mtDNA from individual cells were isolated within porous structure of agarose beads after the agarose gelation and cell lysis.A microfluidic droplet generator was used to form a dense porous structure enough to retain mtDNA from a single cell, and the retention ratio of mtDNA was evaluated ~95% by a qPCR (Figure 2A,B).To mitigate the hindered throughput of agarose droplet generation due to its high viscosity, we incorporated parallelized microfluidic droplet generators which achieved throughput >70,000 drops/min (Figure 2C).mtDNA from single cells within agarose beads were analyzed by using a rolling circle amplification (RCA) (Figure 3A).RCA can localize amplified signal as a few hundred nm to a few m punctate, which are large enough to be retained in agarose bead.First, padlock probe was annealed to a specific region of mtDNA after the denaturation using 60% DMSO.Subsequently, padlock probe was ligated to form a circular DNA and primer was hybridized on the circle DNA.After RCA reaction, RCA products, large punctate were generated within agarose beads and visualized by fluorescent detection probes.Based on this method, Cross-contamination of mtDNA between beads during a sample preparation was validated by using mouse and human cells (Figure 3B).There was minimal cross-contamination by noting that agarose beads have either ATTO565 or ATTO647 fluorescence signal, which was confirmed by a flow cytometer (Figure 3C,D).Agarose beads that had encapsulated single cell was ensured by SYBR green gDNA stain.Fluorescence signal of RCA products generated from mtDNA showed significant difference between SYBR green positive and negative beads.Then we evaluated multiplex assay on single-mtDNA from single cell to analyze large area deletion by targeting D-loop and 4,977 deletion regions (Figure 4A).RCA products labelled with different fluorescence were distributed throughout a single agarose bead (Figure 4B,C).Different fluorescence profile of each bead analyzed by imaging and flow cytometry, which revealed heteroplasmy and intercellular heterogeneity of mtDNA 4,977 deletion (Figure 4D,E).Furthermore, padlock probe-based RCA assay was applied for mtDNA single nucleotide variation (SNV) analysis in single-cell level (Figure 5A).By locating SNV region at the end of padlock probe, SNV of mtDNA can be detected by RCA products labelled with different fluorescent detection probes.It was shown that more RCA products were generated from padlock probes targeting wild type in both single-plex and multiplex assay (Figure 5B).The proposed method will open a new avenue for advancing our understanding of the biological role of mtDNA heteroplasmy.