Nt bbvci

Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA). E. coli DNA topoisomerase I was purified as described previously22 (link). The following synthetic oligonucleotides were purchased from MWG-Biotech, Inc. (Huntsville, AL): FL882 (5′-CCCTCAGCCCGACAGCACGAGACGATATATATATATATATATATATATATATATATATATATATATGGGCCAACCAACCAGCCCCTCAGCG-3′), FL883 (5′-GATCCGCTGAGGGGCTGGTTGGTTGGCCCATATATATATATATATATATATATATATATATATATATATATCGTCTCGTGCTGTCGGGCTGAGGGCATG-3′), FL905 (5′-TCAGCCCGACAGCACGAGACGATATATA[Dab-dT]ATATATATATATATATATATATATA[Fl-dT]ATATATATGGGCCAACCAACCAGCCCC-3′), FL919 (5′-TCAGCCCGACAGCACGAGACGATATA[Dab-dT]ATATATATATATATATATATATATATATA[Fl-dT]ATATATGGGCCAACCAACCAGCCCC-3′), FL920 (5′-TCAGCCCGACAGCACGAGACGATATATATA[Dab-dT]ATATATATATATATATATATA[Fl-dT]ATATATATATGGGCCAACCAACCAGCCCC-3′), and FL924 (5′-TCAGCCCGACAGCACGAGACGATATATA[BHQ2-dT]ATATATATATATATATATATATATA[TAM-dT]ATATATATGGGCCAACCAACCAGCCCC-3′) where Dab-dT, Fl-dT, BHQ2-dT, and TAM-dT represent dabcyl-dT, fluorescein-dT, BHQ2-dT, and TAMRA-dT, respectively. QIAquick Nucleotide Removal Kit and QIAquick Gel Extraction Kit were obtained from Qiagen, Inc (Valencia, CA).

To obtain an improved SO reference genome, we generated a hybrid (10xG and Bionano Genomics) assembly following the approach in Levy-Sakin et al. (2019) (link). Briefly, we obtained high-molecular-weight DNA from blood sample of Sequoia and used this to generate a 10xG linked-read library (using their Chromium system) and Bionano genome maps (using their Irys system). Instead of generating a single-genome map with the enzyme Nt.BspQI, we generated two sets of Bionano genome maps with the enzymes Nt.BspQI (New England Biolabs [NEB], Ipswich, MA, USA) and Nt.BbvCI (NEB, Ipswich, MA, USA). The 10xG library was sequenced to an average depth of approximately 60× and assembled using Supernova v1.1 (Weisenfeld et al. 2017 (link)). We then generated hybrid scaffolds using the Bionano genome maps to bridge Supernova scaffolds (see Levy-Sakin et al. 2019 (link) for further details).

Four different DNA tetrahedra extending with the corresponding
DNAzyme subunits were prepared. Then equal amounts of these tetrahedron
structures were mixed (0.5 μM each, 150 μL) with the two
duplexes L₁I₁, L₂I₂ (0.5
μM each), 0.068 μM nicking enzyme (Nt.BbvCI, New England
BioLabs Inc.), the substrates (3 μM S₁, 2 μM
S₂) and with or without variable concentrations of the
corresponding inhibitor strands, B₁ and B₂.
The prepared mixtures were added with the fuel strands, L₁′ and L₂′ (2 μM each) and time-dependent
fluorescence changes were monitored spectroscopically at 25 °C.

Equal
amounts of four different tetrahedron structures were mixed (1 μM
each, 150 μL) with the two duplexes L₁I₁, L₂I₂ (1 μM each), and 0.136 μM
nicking enzyme (Nt.BbvCI, New England BioLabs Inc.) and with or without
different concentrations of the corresponding inhibitor strands, B₁ and B₂. The prepared mixtures were added with
the fuel strands L₁′ and L₂′ (4
μM each) and time-dependent fluorescence changes were monitored
spectroscopically at 33 °C. The time-dependent fluorescence changes
corresponding to the two tetrahedra dimers are followed by the evaluation
of the FRET signals of fluorophore pairs of Cy3/Cy5 and FAM/TAMRA
associated with the two tetrahedra dimers. Using the respective calibration
curves of the two pairs of chromophores, Figures S3 and S7, the transient FRET signals were translated to transient
concentration changes of the tetrahedra dimers T₁/T₂ and T₃/T₄. It should be noted, however,
that the FRET signals of Cy3/Cy5 and FAM/TAMRA exhibit overlap features.
To overcome this difficulty, each of the gating states shown in Figure 4 was characterized
in two-separate analysis samples where one sample included the T₁/T₂ tetrahedra dimer labeled with Cy3/Cy5 and the
other tetrahedra constituent, T₃/T₄, was nonlabeled.
The second analysis sample included nonlabeled T₁/T₂ and the FAM/TAMRA-labeled T₃/T₄ constituent.