Present techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ~300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ~40 picometer sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.
Supplementary Figure 1: CRISPRa at the endogenous gene target ldhA does not follow predicted trends.Eight scRNA target sites (L1-L8) upstream of the ldhA promoter were selected. Three of the target sites (L5-L7) were within the 40 bp window where CRISPRa is effective (-100 to -60). While L1, L4, and L5 resulted in weak increases in gene expression, there was no apparent relationship between the position of the sites and ldhA expression levels. Gene expression was measured using RT-qPCR. Fold activation represents expression levels relative to an off-target control (hAAVS1). Bars indicate the average values between three technical replicates and black dots indicate the values of individual replicates. Error bars indicate the standard error of the mean between three technical replicates. S3 Supplementary Figure 2: CRISPRa activity depends on the target sequence on the scRNA. Reporter cassettes that differ only by the sequence of the 20 base scRNA target site give a broad range of gene expression levels, demonstrating that the sequence of the scRNA target site can have a substantial effect on CRISPRa. Three new reporter plasmids were constructed where the J306 target site, located at -81 from the TSS, on the J3-J23117-mRFP1 reporter was replaced by the J104, J106, and J108 sequence. Activation at each promoter was tested when CRISPRa was targeted to their cognate scRNA site. The off-target negative control (OT) represents a strain expressing the original reporter with the J306 site and the CRISPRa components to target an offtarget site (J206). Fluorescence/OD 600 values were measured using a plate reader. Bars indicate the average values between three biological replicates. Black dots indicate the values of individual biological replicates. Error bars indicate the standard deviation between biological replicates. S4 S5 Supplementary Figure 3: The sharp positioning dependence of CRISPRa is observed across multiple promoters. A) The sharp positioning requirements of CRISPRa are not significantly affected by the location or composition of the inserted sequence. Reporters were based on the J1-J23117-mRFP1 (Supplementary Figure 3A) with base shifts introduced in different ways. In the J1 reporter A, bases were inserted upstream of the -35 region. In the J1 reporter B, a different sequence was
Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.
Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.
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