Nanomanipulation of Single RNA Molecules by Optical Tweezers
August 20th, 2014
Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.
The overall goal of the following experiment is to demonstrate a single molecule mechanical unfolding experiment on an RNA structure. In the first step, a flow chamber is constructed and mounted to the instrument. In the second step, micron size beads are mixed with the RNA sample of interest, and then the two different sizes of beads are loaded into the flow chamber.
Finally, the flow chamber and the optical trap can be used to steer the beads into contact with one another to evaluate a variety of single molecule interactions. Ultimately, this nano manipulation technique can be used to study the folding of RNA at the secondary and tertiary structural levels. This method can help answer key questions in the RNA folding field, such as how does an RNA fold and unfold?
How does it transit between different confirmations in response to an environmental change? And how does it interact with other molecules? Today's experiment will be performed by my graduate student, Mr.Williams Stevenson, To make a flow chamber begin by drilling six two millimeter diameter holes into one number two cover slip.
Then use a laser engraver to cut three two millimeter wide slits into two pieces of double-sided polyamide tape. Attach one of the pieces of tape to the cover slip, making sure to align the holes in the tape with the holes in the cover slip. Attach the other piece of tape to a clean un drilled cover slip.
Remove the protective covering from both pieces of tape, and then place a glass pulled micro pipette and two bi tubes between each of the channels on one of the cover slips. Next sandwich, the two cover glasses, keeping the micro pipette and bypass tubes in place with the two layers of tape. To make a flow chamber, mount the flow chamber onto the backside of a metal frame, matching the holes in the chamber over the fluidic channels.
Then turn the frame over and connect the channels to 10 milliliter syringes filled with a buffer of choice via polyethylene tubing. Now mount the entire chamber onto the optical tweezers between the two objectives, ensuring that the front side of the chamber with the fluidic channels faces to the right. Then retract the left objective and plug the brass pins of the frame into the mounting holes on the tweezers and tighten the screws to fix the chamber into position.
Use a bungee cord to lift the instrument off the table. Enclose the instrument in an acoustic box before delivering beads to the reaction site of the flow chamber. First, mix an A kneeled RNA sample with anti-D, dig oxygen ENC coated beads or dig beads, and then incubate the mixture at room temperature for 15 minutes.
During the incubation, lotus suspension of stripped habit and coated beads into a one milliliter syringe, and connect the syringe to the fluidic tubing leading to the bottom channel of the flow chamber. Plunge the syringe to load the strep beads into the chamber, and then use the motor control software to place the optical trap near the opening of the bottom bypass tube and move the entire chamber. Then manipulate the track ball to operate the optical trap and capture a strep bead.
Use the motor control software to move the bead close to the tip of the micro pipette. Next, pull the syringe connected to the micro pipette to vacuum the bead onto the micro pipette, and then gently plunge the syringe to apply flow to the middle channel and flush away any remaining strep beads. Deliver the dig bead sample mixture to the top channel of the chamber and capture a dig bead as just demonstrated.
Then use the optical trap to bring the dig bead close to the strep bead. Apply flow to clean the middle channel. Then to fish a single molecule tethering between the pair of beads.
Place the captured dig bead vertically on top of the strep bead. Next, steer the trap to move the dig bead down towards the strep bead and open a force distance plot window on the monitor to visualize the force changes. Finally, upon contact, move the dig bead vertically away from the strep bead tracking the force of their separation.
Once an RNA molecule is tethered between beads by its five prime and three prime ends, it can be repeatedly stretched and relaxed. Elasticity of the double stranded handles is indicated by non-linear force extension curves. Cooperative folding and unfolding of the RNA structure between the handles are reflected by abrupt rips and zips in the force extension curve with negative slopes, the refolding of the hairpin is indicated by a zip on the force extension curve.
Importantly, the same molecule may be unfolded and refolded at different forces each time. Such a trend is evident in the distributions of the transition forces. A feedback mechanism can be used to maintain a constant force on the RN.A hairpin folding of an RNA hairpin is indicated by a shortening of extension, whereas unfolding is reflected by an increase in extension under the passive mode, the positions of the trap and the micro pipette are both fixed.
When an RNA folds into a hairpin and shortens its extension, the trapped bead moves away from the center of the trap increasing force. When the RNA is extended to a single strand, the bead moves towards the trap center decreasing force. Using this technique, RNA folding pathways can be observed and manipulated.
With pico Newton and nanometer resolution state lifetimes and transition forces of single molecules can be amassed to derive rate constants and free energies of RNA folding. While attempting this procedure, it's important to remember to be patient while phishing a single molecule. The chance to find a single molecule tether between two beads is very low.
Therefore, many pairs of beads may not stick together at all.
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Overview
This article describes the use of optical tweezers to study RNA folding by manipulating individual RNA molecules. The procedures for synthesizing RNA, calibrating the instrument, and manipulating single molecules are outlined.
Key Study Components
Area of Science
- Biophysics
- Molecular Biology
- RNA Folding
Background
- Optical tweezers allow for the manipulation of single molecules.
- Understanding RNA folding is crucial for insights into its function.
- Mechanical unfolding experiments can reveal structural dynamics.
- Single molecule techniques provide detailed information on molecular interactions.
Purpose of Study
- To demonstrate mechanical unfolding of RNA structures.
- To evaluate single molecule interactions using optical traps.
- To answer fundamental questions about RNA folding and unfolding.
Methods Used
- Construction of a flow chamber for the experiment.
- Mixing micron-sized beads with RNA samples.
- Loading beads into the flow chamber for manipulation.
- Using optical traps to steer beads into contact for interaction evaluation.
Main Results
- Successful demonstration of RNA folding and unfolding mechanics.
- Evaluation of interactions between RNA and beads.
- Insights into the secondary and tertiary structures of RNA.
- Establishment of a method for studying RNA dynamics.
Conclusions
- The optical tweezing technique is effective for RNA studies.
- This method can help address key questions in RNA folding.
- Future studies can build on these findings to explore RNA behavior.
Frequently Asked Questions
Executive Industry Relevance
Single-molecule nanomanipulation of RNA using optical tweezers enables direct interrogation of RNA folding pathways and structural transitions, addressing a critical challenge in understanding RNA function beyond sequence analysis. This capability enhances predictive confidence in target validation and de-risks early discovery by providing quantitative, molecule-specific insights into RNA structural dynamics. The approach supports portfolio decisions by clarifying mechanistic underpinnings of RNA-based targets and regulatory elements.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct measurement of RNA folding and unfolding transitions at the single-molecule level.
- Clarifies the structural basis of RNA regulatory function, supporting mechanistic de-risking.
- Provides quantitative force-extension data to inform target validation and triage.
Screening & Assay Development
- Establishes validated single-molecule systems for downstream biophysical assays.
- Supports reproducible measurement of RNA conformational changes under controlled conditions.
- Facilitates assay standardization by enabling repeated manipulation of the same molecule.
Translational & Preclinical Research
- Aligns RNA structural interrogation with disease-relevant regulatory mechanisms when applicable.
- Enables continuity from discovery to preclinical validation for RNA-targeted modalities.
- Supports risk-adjusted advancement by providing mechanistic evidence of RNA behavior.
Pipeline & Workflow Integration
This nanomanipulation method integrates into the discovery continuum from early hypothesis testing through lead identification for RNA-centric targets.
- Discovery Biology: Supports hypothesis testing by revealing real-time RNA folding pathways and conformational transitions.
- Screening: Provides reproducible, quantitative force-extension outputs for comparative analysis of RNA constructs.
- Analytics: Delivers high-resolution measurements of transition forces, state lifetimes, and folding kinetics.
- Translational Research: Offers mechanistic continuity for RNA-based targets across discovery and preclinical stages.
- Enterprise Reuse: Establishes a reusable platform for diverse RNA structural and interaction studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces ambiguity in RNA target validation.
- Operational Value: Enables standardized, reproducible single-molecule measurements at scale.
- Strategic Value: Informs go/no-go decisions by providing direct evidence of RNA structural behavior.
- Portfolio Impact: Supports risk-adjusted prioritization of RNA-targeted programs.
Implementation Considerations
- Requires expertise in single-molecule biophysics and RNA chemistry.
- Demands specialized instrumentation, including optical tweezers and microfluidic flow chambers.
- Necessitates rigorous calibration and cross-team standardization for reproducibility.
- Adaptation may be needed for different RNA constructs or structural domains.
- Low probability of single-molecule tethering increases experimental time and resource requirements.
