Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion.

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Optical tweezers are essential single-molecule tools for biophysical studies.
• They enable simultaneous measurement of force and torque, offering insights into protein-DNA interactions.
• A novel DNA supercoiling assay using angular optical tweezers (AOT) has emerged for studying transcription-related torque generation.

Purpose of the Study:

• To provide a detailed and practical guide for performing the DNA supercoiling assay using AOT.
• To present protocols for instrument construction, calibration, DNA template preparation, and data analysis.
• To adapt these methods for studying bacterial RNA polymerase (RNAP) and other DNA-based motor proteins.

Main Methods:

• Construction and calibration of an angular optical tweezers (AOT) instrument.
• Preparation of DNA templates for transcription assays.
• Real-time data acquisition and analysis of transcription under DNA supercoiling using E. coli RNAP.

Main Results:

• Demonstration of a practical protocol for AOT-based DNA supercoiling assays.
• Successful application to study torque generation in bacterial RNA polymerase (RNAP).
• Establishment of a versatile method adaptable to other DNA motor proteins.

Conclusions:

• The developed AOT assay provides a powerful method for investigating torque generation during transcription.
• This technique offers novel insights into the mechanics of DNA-based motor proteins.
• The protocols are readily adaptable for diverse applications in molecular biophysics and biochemistry.