Claret Bioscience LLC

A Rapid Method for Global and Targeted Mirna Next Generation Sequencing - Project Summary Micrornas (MIRNAS) Are Small Non-coding Rnas (15-25 NT) That Play a Pivotal Role in the Regulation of Protein Synthesis by Modulating Mrna Translation. Mirna Expression, Localization and Secretion Are Closely Modulated in Response to Biological Stimuli. These Processes Are Often Dysregulated in Cancer Resulting in Mirna Expression Profiles That Are Unique to Cancer Type and Stage. Apart From Its Utility in Early Cancer Detection, Tumor Mirna Profiling Has Been Shown to Improve Patient Survival When Used in Treatment Response Monitoring. Recent Liquid Biopsy Studies Have Demonstrated the Utility of Mirnas as Circulating Biomarkers for Early Cancer Diagnosis and for Monitoring Disease Progression and Treatment Response. However, Existing Assays Are Not Sensitive to the Subtle Changes in Mirna Profiles and Require Prior Knowledge of the Dysfunctional Mirnas. While Next-generation Sequencing (NGS) Can Be Used for Agnostic Mirna Profiling, There Is a Clear Lack of a Rapid, Easily Administrable Ngs-based Molecular Diagnostic Assay That Efficiently Captures Subtle Changes in Microrna Profiles During Disease Progression Owing to the Small Size and Scarcity of Mirna in Biological Samples, Particularly in Biological Fluids. in This Application We Propose to Develop a Novel Approach Called Mireally That Directly Converts Mirna to Sequence-ready Libraries in Just Two Steps – Direct Ligation of Claretbio's Proprietary Adapters to Mirna, Followed by a Step That Performs Reverse- Transcription and Library-amplification in a Single Reaction. the Proof of Concept Work Described Here Will Identify the Ideal Combination of Reagents and Enzymes That Facilitate Efficient Library Conversion, With Metrics That Outperform Current Small-rnaseq Methodologies. in Aim 2 We Will Further Optimize the Protocol and Determine the Limit of Detection and Input Range to Expand the Utility of the Approach With Biological Samples Using Non- Small Cell Lung Cancer as a Model. Finally in Aim 3 We Will Leverage the Unique Design of the Adapters for Targeted Enrichment of Mirna – by Designing Splint Adapters With Sequence Complementarity to the Ends of a Specific Mirna From Complex Mirna Mixtures. This Allows Capture of a Panel of Known Mirna and Their Variants in a Single Workflow, Eliminating the Need for Additional Targeted Enrichment Steps. Accomplishing the Aims Described Here Would Result in a Robust Mirna NGS Assay That Not Only Enables Global Mirna Profiling But Also Helps Monitor Changes to Levels of Specific Mirna Biomarkers and Their Variants Thereby Improving Our Understanding of Intracellular and Circulating Mirna Biology and Facilitating More Precise Disease Diagnosis and Monitoring in the Clinical Setting.