Rolling circle RNA synthesis catalyzed by RNA
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, United Kingdom
- Department of Physics, University of York, United Kingdom
Figures
Figure 1 with 1 supplement
Primer extension on circular RNA (circRNA) templates.
(A) Schematic illustration of rolling circle synthesis (RCS). Red product RNA strand is extended by a triplet at the 3′-end while three base pairs dissociate at the 5′-end keeping the total …
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Figure 1—source data 1
Gel images and numerical values.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig1-data1-v3.zip
Figure 1—figure supplement 1
Purification of circularized RNA (scGAA8-16).
Identification and dissection of circularized RNA were performed by denaturing PAGE. Representative SyBr Gold stained 10% Urea PAGE gel is shown here for illustrating the circularization process of …
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Figure 1—figure supplement 1—source data 1
Gel images.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig1-figsupp1-data1-v3.zip
Figure 2 with 1 supplement
Full-length and beyond full-length RNA-catalyzed RNA synthesis on circular RNA templates.
(A) Product strand of primer extension experiments with primer P10 (red) and eight triplet scRNA template strands. Potential beyond full-length circle synthesis triplets are shaded opaque. (B) …
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Figure 2—source data 1
Gel images and numeric values.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig2-data1-v3.zip
Figure 2—figure supplement 1
Optimization of rolling circle synthesis.
(A) Comparison between linear and circular primer extension using the CHES reaction buffer system (similar to main text Figure 1D that is in the Tris buffer system). Extensions performed at −7°C for …
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Figure 2—figure supplement 1—source data 1
Gel images and numeric values.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig2-figsupp1-data1-v3.zip
Figure 3 with 6 supplements
Molecular dynamics simulation of small circular RNA.
(A) Main conformations (and zoom-in to relevant regions [squares]) observed from simulations in 100 mM MgCl2 on scRNA exploring consecutive states of primer extension, from 9 to 30 bp dsRNA with …
Figure 3—figure supplement 1
Time sequence of snapshots from the simulation of scRNA with 27 bp dsRNA in 100 mM MgCl2 with pyrimidine (template) strand (UUC)12 (khaki), purine (product) strand (GAA) (light blue).
We observe a series of melting and reannealing events in the 3′-end (purple and light green) during the first half of the simulation and in the 5′-end (dark blue and dark green) during the second …
Figure 3—figure supplement 2
Time sequence of snapshots from the simulation of scRNA with 30 bp dsRNA in 100 mM MgCl2.
We observe a series of melting and reannealing events in the 5′-end (dark blue and dark green). The rest of the color code is the same as Figure 3—figure supplement 1.
Figure 3—figure supplement 3
Percentage of frames from the last 100 ns of the simulations presenting canonical hydrogen bond pairing for each base pair (bp).
(A) Linear RNAs solvated with the three buffers (100 mM KCl, 200 mM MgCl2, and 500 mM MgCl2). (B) Rolling circle RNA synthesis (RCS) simulations solvated with 100 mM KCl. (C) RCS simulations …
Figure 3—figure supplement 4
Roll, slide, twist, and major and minor groove widths.
(A–E) Averages and standard deviations (as error bars) of bp-step parameters (roll, slide, and twist) together with major and minor groove widths (MajW and MinW, respectively) calculated over the …
Figure 3—figure supplement 5
Counterion-density maps around RNA molecules that show an occupancy ~10 times or greater than the bulk concentration (in red) as seen in simulations.
These areas are the molecular regions where cations localize preferentially. In the case of 200 mM KCl, these align along the grooves, whereas, in the case of MgCl2, they tend to be closer to the …
Figure 3—figure supplement 6
Averages and standard deviation of RDF of cations around RNA backbone phosphates.
(Top) Averages and standard deviations (as error bars) of radial distribution functions (RDFs) of cations around RNA backbone phosphates. The RDFs indicate the probability of finding an ion within a …
Figure 4 with 2 supplements
RNA-catalyzed RNA synthesis beyond full length for circular templates.
(A) Product strands of primer extension experiments with linear and scRNA templates A–D with primer P91. Opaque sequence illustrate potential beyond full-length synthesis on scRNA. Barcode triplets …
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Figure 4—source data 1
Full analysis of sequencing data used in Figure 4B.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig4-data1-v3.zip
Figure 4—figure supplement 1
Deep sequencing of extension products.
(A) Representative SyBr Gold stained 10% Urea PAGE gel showing linear and circularized templates A–D. Circularized RNA was retained in the gel as expected. A small degree of hydrolyzed RNA is seen …
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Figure 4—figure supplement 1—source data 1
Gel images.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig4-figsupp1-data1-v3.zip
Figure 4—figure supplement 2
Controls for deep-sequencing data.
(A) Plot shows the fidelity ratio at the noted triplet positions between extension reactions where the templates were incubated either in one-pot (Mix) or in individual tubes (Ind). This is shown …
Figure 5 with 1 supplement
RNA-catalyzed branched RCS.
(A) Product strand of primer extension experiments with scRNA template containing two priming sites (I and II) for primer P91. Depending on the priming site two different products will be made (I or …
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Figure 5—source data 1
Gel images and full data analysis of sequencing data used in Figure 5D.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig5-data1-v3.zip
Figure 5—figure supplement 1
10% Urea PAGE separation of circular template extension reaction used for deep sequencing.
Excised gel piece is marked with green.
Figure 6 with 1 supplement
Steps of a viroid-like replication cycle catalyzed by RNA alone.
(A) Illustration of the µHHz (−) and (+) strand. (B) Schematic illustration of the RNA-catalyzed viroid-like replication with steps comprising RNA-catalyzed combined RCS (steps 1–3), resection …
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Figure 6—source data 1
Gel images.
- https://cdn.elifesciences.org/articles/75186/elife-75186-fig6-data1-v3.zip
Figure 6—figure supplement 1
Kinetic analysis of the µHHrz.
(A) Quantification of band intensities as a function of time. Here, we see that cut RNA accumulates while the amount of circular RNA seems to reach an equilibrium. (B) Fraction of circular RNA …
Videos
Video 1
Movie of the RCS simulation where dsRNA is 27-bp long.
We observe fraying and annealing of 5′- and 3′-ends demonstrating the quick timescales of these transitions.
Video 2
Movie of the RCS simulation where dsRNA is 30-bp long.
We observe again fraying and annealing of 5′- and 3′-ends demonstrating the quick timescales of these transitions.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75186/elife-75186-transrepform1-v3.pdf
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Supplementary file 1
Oligonucleotide sequences.
- https://cdn.elifesciences.org/articles/75186/elife-75186-supp1-v3.docx
