Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS)

DNA or RNA templated reactions are attractive for nucleic acid sensing and imaging. As for any hybridization-based sensing, there is a tradeoff between sensitivity (detection threshold) and resolution (single nucleotide discrimination). Longer probes afford better sensitivity but compromise single nucleotide resolution due to the small thermodynamic penalty of a single mismatch. Herein we report a design that overcomes this tradeoff. The reaction is leveraged on the hybridization of a minimal substrate (covering 4 nucleotides) which is confined by two guide DNAs functionalized respectively with a ruthenium photocatalyst. The use of a catalytic reaction is essential to bypass the exchange of guide DNAs while achieving signal amplification through substrate turnover. The guide DNAs restrain the reaction to a unique site and enhance the hybridization of short substrates by providing two π-stacking interactions. The reaction was shown to enable the detection of SNPs and SNVs down to 50 pM with a discrimination factor ranging from 24 to 309 (median 82, 27 examples from 3 oncogenes). The clinical diagnostic potential of the technology was demonstrated with the analysis of RAS amplicons obtained directly from cell culture.


Total RNA extraction from cells and RT-PCR S4
1.4 Information of sequences (Table S1)  Detection limit of CHOMS templated reactions using RuUD1, RuDD1, DC1 S10 Figure S6 CHOMS templated reaction for selective discrimination of SNVs S11 Figure S7 CHOMS templated reaction using DC1-A, RuUD1, RuDD1, and KRAS-T S12 Figure S8 Two-color system of CHOMS templated reactions S13 Figure S9 Analysis of PCR products by 10 % 8M urea denaturing polyacrylamide gel electrophoresis S14 Figure S10 Two-color CHOMS templated reaction using RT-PCR products S15 Table S2 Discrimination factor and two sample unpaired t-test S16 3. Mass spectra of the synthesized DNAs and PNAs S17

References S40
Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2020 S2
Each experiment was performed in triplicates. Stock solutions of each reaction component were prepared in water at 0.5 µM. In a plastic 96-well plate (standard opaque), 50 µL of pH 7.4 4×PBS buffer, 10 µL of 0.2 % Tween-20, 10 µL of 100 mM sodium ascorbate, and 2 or 3 µL of 100 ng/µL single stranded sperm DNA were added to the reaction well. To the mixture, the stock solution of RuUD, RuDD, target SNV sequence (or PCR amplicons), and 4-mer coumarin or rhoamine PNA (DC or DR) were added to the desired concentration (final volume 200 µL). Water was added to reach the 200 µL of reaction volume.
Final mixtures were mixed well using a micropipette and directly applied to templated reaction without S4 further manipulation.
In case of low abundant detection experiment, 0.5 µM stock solutions of wild type and mutant type were mixed in different ratio (0 % to 100 %) and then 2 µL of the resulting mixture was used for the templated reaction (5 nM of total target concentration at 200 µL volume).
Fluorescence of the samples was measured immediately after irradiation. Molecular Devices Spectra Max M5 were used for measurement of fluorescence intensities with following parameters (For coumarin: λex: 360 nm, λem: 460 nm, cutoff: 455 nm, PMT gain: medium, flash per read: 6, shake 5 sec before first read; for rhodamine: λex: 490 nm, λem: 530 nm, cutoff: 515 nm, PMT gain: medium, flash per read: 6, shake 5 sec before first read). The well-plate was irradiated with a collimated LED light 10 cm above the plate (455 nm, 1W: Thorlabs, part number M455L2-C1 -www.thorlabs.com). The percentage of conversion was calculated based on a titration curve of coumarin or rhodamine. We validated that the reaction reaches a plateau and that the fluorescent units of this plateau indeed correspond to 100% yield according to the titration curve.
For calculation of discrimination factor (DF), we adopted the equation, DF = (signal of WT targetsignal of control (no target)) / (signal of SNV target -signal of control (no target)), using the Fl. values obtained at 60 min. of reaction time.

Total RNA extraction from cells and RT-PCR
Total RNA extraction from each HT-29, SW620, A549 cell line was done by RNeasy Mini Kit (QIAGEN) using 5×10 6 cells. 5 µg of total RNA was converted to cDNA of total mRNA by using oligo(dT)15 of Reverse Transcription System (Promega). The mixture of cDNA was directly used for PCR using conditions: 1 µL of aliquot of cDNA mixture, 5 µL of 5×Phusion HF buffer (ThermoScientific), 0.5 µL of 10 mM dNTP mix (ThermoScientific), 1 µL of 10 µM forward primer (FP),  Ru

Information of sequences
Ru-ATACTCCATAATTTAAAA RuDD3 ATTCTCGTCTCCACAG   and RuDD1, 20 µL of PCR sample, total 200 µL. Table S2. Discrimination factor and two sample unpaired t-test with unequal variances (p-value) between target and single mismatch sequences. The target signal was below background (none) signal for #16, 19, 21. The discrimination factor is calculated as follow: (fluorescence of target-background)/( fluorescence of mismatch-background); The two sample t-test was calculated from raw fluorescence value using ORIGIN software. The discrimination for two color is calculated as follow: (