INTRODUCTION
1 Optical tweezers principle and setup. A Single light beams gradient trap. B A dual trap optical tweezers diagram. C House-made optical tweezers set up in Dr. Guo’s lab at the Ohio State University (Ghimire et al. 2020). D Schematic showing a biomolecule tethered in a dual trap optical tweezers |
2 Total internal reflection fluorescence setup for single molecule fluorescence microscopy. A Prism based TIRF schematic diagram. B Prism based TIRF set up in Dr. Guo lab at the Ohio State University (Shu et al. 2007; Zhang et al. 2007, 2010a, 2010b). C Imaging of RNA dimers composed of one Cy3-RNA (green) and one Cy5 RNA (red). Yellow color indicates the overlap of Cy3 and Cy3, suggesting the formation of dimers containing both Cy3 and Cy5. D A typical fluorescence image of individual phi29 DNA packaging motor, showing six photobleaching steps, indicating that there are six copies of pRNA molecules on the DNA packaging motor |
3 Construction and characterization of different RNA nanoparticles based on 3WJ. Angles of original 3WJ were stretched to accommodate different shapes of 2D or 3D RNA nanoparticles. In each of the structure, schematic and AFM/Cryo-EM images are shown. The 97° interior angle of the 3WJ was either compressed to 60° (left column), or stretched to 90° (center column) and 108° (right column), proving the rubbery property of RNA nanostructure. The scale bar is 10 nm in each image. Adapted from Ghimire et al. (2020) with permission from American Chemical Society |
OPTICAL TWEEZERS
General principle of optical tweezers
Optical tweezer to elucidate the elasticity of RNA and DNA
4 Stretching DNA with optical tweezers. A Experimental schematic showing DNA is anchored to the cover glass by a stalled RNA polymerase complex while the other end is attached to a bead held in the optical trap. B Force-extension curve of a single DNA molecule. Adapted from Wang et al. (1997) with permission from Elsevier |
Optical tweezers to quantify the forces in base paring
5 Unzipping DNA with optical tweezers. A Schematic of unzipping of DNA experiment. B Force versus extension curves corresponding to mechanical unzipping of a single lambda-phage DNA molecule. C The measured (bottom) and the calculated (top) curve for force vs extension curve for DNA. Adapted from Bockelmann et al. (2002) with permission from Elsevier |
Optical tweezers to study unfolding of RNA nanostructures
Optical tweezers to study unfolding of DNA nanostructures
7 Unfolding of DNA G-quadruplex with optical tweezers. A Schematic of experimental design. B Rupture force histogram for the unfolding events. Adapted from Yu et al. (2009) with permission from American Chemical Society |
Optical tweezer to elucidate the elasticity of RNA nanoparticles
8 Rubbery property of RNA square as demonstrated by optical tweezer analysis. A Schematic diagram of dual trap optical tweezers with a tethered RNA construct sandwiched between two dsDNA handles via affinity linkers. B A typical force-extension curve for stretching (red) and relaxing (blue) of square nanoparticle. Inset shows magnified view of conformational change of the square nanoparticle. The 50 nm scale bar is for the x-axis of force extension curve. Adapted from Ghimire et al. (2020) with permission from American Chemical Society |
1 Extension change (∆x) corresponding to force (Force) |
Force (pN) | ∆x (nm) | kcc (s−1) | x† (nm) | ∆G† (kcal/mol) | |
The value for DNA and RNA concerning conformation change rate constant (kcc), distance to the transition state (x†) and conformation change energy barrier (∆G†) are shown. Adapted from Ghimire et al. (2020) with permission from American Chemical Society | |||||
DNA | 44.2 ± 2.3 | 2.99 ± 0.03 | 0.0016 ± 0.0007 | 0.132 ± 0.008 | 13.5 ± 0.5 |
RNA | 44.6 ± 4.1 | 4.1 ± 0.10 | 0.005 ± 0.0030 | 0.10 ± 0.020 | 11.4 ± 1.4 |
The optical tweezer elucidated rubber property of RNA nanoparticles supported by in vivo imaging technology
9 Demonstration of the rubbery property of RNA nanoparticles by comparing the retention time in tumor, kidney, and liver. A The Cy5.5 labeled nanoparticles were detected by ex vivo organ 8 h post-injection in mice bearing KB xenograft (T: tumor, H: heart, S: spleen, Lu: lung, K: kidney, and Li: liver; Color scale: radiant efficiency (p⋅s−1⋅cm−2⋅sr−1) / (μW⋅cm−2)). B Quantitative analysis of whole body biodistribution to quantify the ratio of tumor to liver and tumor to kidney using images from A. C Quantitative analysis of biodistribution in tumor to liver and tumor to kidney ratio, quantified from the homogenized organ sample. Adapted from Ghimire et al. (2020) with permission from American Chemical Society |
10 Demonstration of the rubbery property of RNA nanoparticles through renal excretion by comparing tumor and kidney retention time using ex vivo organ images. The near-infrared AF647 labelled 10 nm and 20 nm squares were detected in the kidney 12 and 24 h post systemic injection. Both the 10 and 20 nm RNA squares were excreted through kidney filtration, while with longer retention time in tumor by comparing the organ intensity after 12 h. Adapted from Ghimire et al. (2020) with permission from American Chemical Society |
11 Demonstration of the rubbery property of dsRNA and RNA nanoparticles in urine sample after 30 min of IV injection. Near-infrared AF647 labelled dsRNA, RNA 3WJ and 4WJ were found in mice urine assayed by 12% native gel 0.5 h post IV injection. Adapted from Ghimire et al. (2020) with permission from American Chemical Society |
SINGLE-MOLECULE TOTAL INTERNAL REFLECTION FLUORESCENCE (SmTIRF)
General principle of smTIRF
Single molecule photo bleaching technology in single molecule counting of RNA
Single fluorophore labeling of RNA
Photobleaching assay and analysis of the photobleaching traces
Counting of pRNA in phi29 DNA packaging motor
Analysis of the photobleaching traces
Counting of pRNA in active packaging intermediates
Simultaneous dual color photobleaching of differently labeled pRNA on phi29 DNA packaging motor
Elucidating mechanism of pRNA ring assembly on the motor
12 Dual color photobleaching of differently labeled pRNAs within the same motor/pRNA complex. A Design of dual-labeled pRNA for motor binding. B Typical overlaid fluorescence image of the dual color labeled motor/pRNA complexes. Green: Cy3; Red: Cy5; Yellow: Cy3/Cy5 overlay. C A dual-color photobleaching trace of motor/pRNA complex showing three Cy3-pRNA molecules and three Cy5-pRNA molecules on the same motor. D Comparison of the experimental histogram of photobleaching steps with the theoretical histograms for Cy3-pRNA based on 70% labeling efficiency. Adapted from Shu et al. (2007) with permission from European Molecular Biology Organization |
Statistical analysis of photobleaching histograms
13 Statistical analysis to obtain the actual copy numbers of pRNA on motor. A Experimental histogram of photobleaching steps of motor/Cy3-pRNA complexes. B Fitting of the experimental data with different statistic models (Models 1–4) that were constructed based on 70% labeling efficiency for Cy3-pRNA. The best fit is the Model 1, indicating the presence of six pRNA molecules on the motor that were assembled from three dimers. Adapted from Shu et al. (2007) with permission from European Molecular Biology Organization |
Single molecule photo bleaching technology in single molecule counting of proteins
14 Single molecule photobleaching for stoichiometry of protein. A Stepwise single-molecule photobleaching of |
Single molecule fluorescence to study folding of RNA structures
15 Real time observation of unfolding the duplex DNA−RNA hybrid and folding of RNA. A FRET trajectories showing the PIFE peaks (blue arrow) and the maiden 2D (green arrow) and 3D (magenta arrow) folding events. B Schematics of the “order-of-events” in A. Adapted from Hua et al. (2018) with permission from American Chemical Society |
COMBINATION OF OTHER METHODS WITH OPTICAL TWEEZERS/smFRET
16 Combination of experiments with high-resolution optical trap and fluorescence (not to scale). A Left panels: polystyrene microspheres (grey) are held in optical traps (orange cones), tethered by an engineered DNA molecule (blue) containing a variable central segment flanked by long double-stranded DNA (dsDNA) handles. Fluorophores are excited by a green laser (green cone). B Time traces showing simultaneous measurement of fluorescence and tether extension by oligonucleotide hybridization with short oligonucleotides (blue line) labeled with a fluorophore (green disk) bind and unbind to a complementary ssDNA section in the center of the tethered DNA. The fluorescence and change in tether extension upon hybridization are recorded simultaneously. Adapted from Chuang et al. (2019) with permission from American Chemical Society |