Ribozymes Methods and Protocols

This volume provides protocols designed to study the function and the structure of diverse ribozymes. Chapters guide readers through different techniques to identify and characterize new ribozymes and methods to use ribozymes to alter the function of CRISPR-based guide RNAs, AgoshRNAs and aptamers or to study RNA capping and long non-coding RNAs. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

 Authoritative and cutting-edge, Ribozymes: Methods and Protocols aims to help in accelerating ribozyme research and inspiring others to develop new methods to study ribozyme structure and function.

Part I: Ribozyme Function

1. Conjugation as a Highly Sensitive Assay to Study Group II Intron Splicing in vivo

Félix LaRoche-Johnston, Caroline Monat, and Benoit Cousineau

2. Co-transcriptional Analysis of Self-cleaving Ribozymes and their Ligand Dependence

Luiz F. M. Passalacqua and Andrej Lupták

Part II: Ribozyme Indentification and Characterization

3. Cloning and Detection of Genomic Retrozymes and their circRNA Intermediates

Amelia Cervera and Marcos de la Peña

4. Demonstration of a Ribozyme in Epsilon Domain of Hepatitis B Virus RNA

Dibyajnan Chakraborty and Sagarmoy Ghosh

5. In vitro Selection of Varkud Satellite Ribozyme Variants that Cleave a Modified Stem-loop Substrate

Pierre Dagenais and Pascale Legault

6. Characterization and Optimization of a Deoxyribozyme with a Short Left Binding Arm

7. Computer-aided Design of Active Pseudoknotted Hammerhead Ribozymes

Sabrine Najeh, Kasra Zandi, Samia Djerroud, Nawwaf Kharma, and Jonathan Perreault

8. Inverse RNA Folding Workflow to Design and Test Ribozymes Including Pseudoknots

Mohammad Kayedkhordeh, Ryota Yamagami, Philip C. Bevilacqua, and David H. Mathews

Part III : Ribozyme Structure

9. Using an L7Ae-tethered, Hydroxyl Radical-Mediated Footprinting Strategy to Identify and Validate Kink-Turns in RNAs

Stella M. Lai and Venkat Gopalan

10. SHAPE Profiling to Probe Group II Intron Conformational Dynamics during Splicing

11. Dynamics-Function Analysis in Catalytic RNA Using NMR Spin Relaxation and Conformationally Restricted Nucleotides

Charles G. Hoogstraten, Montserrat Terrazas, Anna Aviñó, Neil A. White, and Minako Sumita

Part IV : Ribozyme Conjugations

12. Design and Evaluation of Guide RNA Transcripts with a 3’-terminal HDV Ribozyme to Enhance CRISPR-based Gene Inactivation

Ben Berkhout, Zongliang Gao and Elena Herrera-Carrillo

13. Design and Evaluation of AgoshRNAs with 3’-terminal HDV Ribozymes to Enhance the Silencing Activity

Ben Berkhout and Elena Herrera-Carrillo

14. Cloning and Detection of Aptamer-ribozyme Conjugations

Ryan P. Goguen, Anne Gatignol, and Robert J. Scarborough

Part V: Ribozyme as Tools to Study other RNAs

15. Use of a Lariat Capping Ribozyme to Study Cap Function in Vivo

Max Pietschmann, Gregor Tempel, Maral Halladjian, Nicolai Krogh, and Henrik Nielsen

16. Long Non-coding RNA Depletion using Self-cleaving Ribozymes

Alex C Tuck and Marc Bühler