M13mp18 single stranded dna

Indium tin oxide-coated glass slides (50 × 24 × 0.175 mm) with an ITO thickness of 17 ± 2 nm and a sheet resistance of 1200 ± 200 Ω/sq were obtained from Hans Tafelmaier Dünnschicht-Technik GmbH (Rosenheim, Germany). Me-O-PEG-(CH2)₃-Si(OMe)₃ with a MW of 460–590 D was bought from ABCR (#SIM6492.7, Karlsruhe, Germany). Avidin-Cy3 conjugate (#A4500-20) was purchased from USBiological (Swampscott, USA). Genomic DNA ULS Labeling Kit (#5190-0419) was supplied by Agilent Technologies (Vienna, Austria). Avidin (#A9275) and Amicon Ultra centrifugal filter untis (#Z648043-24EA) were obtained from Sigma-Aldrich Handels GmbH (Vienna, Austria). GelRed™ Nucleic Acid Gel Stain (#41003) was supplied by VWR International and Agarose NEEO ultra-quality (#2267.2) was purchased by Carl Roth GmbH (Karlsruhe, Germany). M13mp18 single-stranded DNA was purchased from NEB (Ipswich, USA). Conjugated DNA strand 5′-Biotin-TEG-/CTC GCT TCT GTC TAT CTT GGC-3′ were synthesized by Integrated DNA Technologies (Leuven, Belgium).

The DNA frame and the Holliday Junction structure were assembled in a 25 μl solution containing 10 nM M13mp18 single-stranded DNA (New England Biolabs), 50 nM staple strands (226 strands), 20 mM Tris–HCl (pH 7.6), 1 mM EDTA, and 10 mM MgCl₂. The mixture was annealed from 85 to 15°C at a rate of −1.0°C/min. The four-way junctions, which contain a 13-bp homologous core, were constructed by annealing four strands HJ₃₆-a, HJ₃₆-b, HJ₃₆-c and HJ₃₆-d (for 36-nt junction) or HJ₄₁-a, HJ₄₁-b, HJ₄₁-c and HJ₄₁-d (for 41-nt junction). The sequences of the oligonucleotides used for constructing four-way junctions are seen in Supplementary Figure S1. The HJ structures were subsequently incorporated into the DNA frame by annealing the mixture from 40 to 15°C at a rate of −0.5°C/min using thermal cycler. The sample was purified by gel-filtration chromatography (GE sephacryl-400, GE Healthcare Japan, Tokyo, Japan) to remove excess amounts of the staple strands and the HJ structures (Supplementary Figure S2).

Cleavage assays were prepared in reaction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 1 mM DTT, 5% glycerol and varying concentration of MgCl₂). The Cas12a-crRNA RNP was first formed by incubating at 37 °C for 10 min at a final concentration of 20 nM Cas12a and 30 nM crRNA. For cleavage assays in which two plasmids were mixed, the empty pUC19 was added to a final concentration of 15 ng/μL and the activator target or plasmid library was added to a final concentration of 1.5 ng/μL. The DNA was preheated at 37 °C for 10 min and then mixed with RNP to initiate cleavage. Aliquots were quenched at the indicated time points by adding an equal volume of phenol-CHCl₃-isoamyl alcohol and analyzed by agarose gel electrophoresis as described above.
The ssDNA collateral cleavage assays were performed similarly, but with the following modifications. The Cas12a-crRNA RNP was incubated with a short dsDNA activator (PS4 sequence from (Murugan et al., 2020 (link)), final concentration 30 nM) at 37 °C for 10 min before being added to M13mp18 single-stranded DNA (New England Biolabs, final concentration 25 ng/μL). Aliquots were quenched at the indicated time points by adding an equal volume of phenol-CHCl₃-isoamyl alcohol and analyzed by agarose gel electrophoresis as described above.

A solution (50 μL) containing M13mp18 single-stranded DNA (New England Biolabs, 10 nM) and staple DNA strands (5 equiv., 50 nM, nucleotide sequences of all staple DNA strands are described in our previous report18 (link) and Table S13†) in buffer (40 mM Tris–HCl, 20 mM acetic acid, 12.5 mM MgCl₂, pH 8.0) was heated at 95 °C for 1 min, annealed at 53 °C for 30 min, and held at 4 °C in a thermal cycler. The samples were purified by size-exclusion chromatography (400 μL volume of Sephacryl S-400, GE Healthcare) equilibrated with a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, and 12.5 mM MgCl₂ in Ultrafree-MC-DV (Millipore).

Reactions were performed at 37°C in a buffer containing 25 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate, 0.01% NP-40-S, 1 mM DTT, 10 mM Mg(OAc)₂, 0.1 mg/ml BSA, 5 mM ATP, 200 μM CTP, GTP, UTP, 30 μM dATP, dCTP, dGTP, dTTP, and 33 nM α-[³²P]-dCTP. 0.5 nM M13mp18 single-stranded DNA (New England Biolabs) was pre-incubated with 40 nM RPA for 10 min. Reactions were initiated by the addition of 20 nM Pol α-primase. After 20 min, reactions were quenched by addition of EDTA to 50 mM. Post reaction processing and analysis were performed as described for budding yeast primase-polymerase assays on M13mp18 ssDNA.

All short staple strands were purchased from Invitrogen (China) and used as received. M13mp18 single stranded DNA was purchased from New England Biolabs. Chemicals were purchased from Sigma-Aldrich. Quantum dots were purchased from Invitrogen (China).