RNA secondary structure ensembles and alternative states play critical roles in RNA function and regulation. Most RNAs can fold into multiple alternative structures, and these ensembles are dynamically regulated by cellular processes. Understanding these ensembles is essential for elucidating RNA biology and developing therapeutic strategies. RNA structural ensembles are influenced by factors such as RNA sequence, post-transcriptional modifications, and interactions with proteins and ions. These ensembles are also critical for RNA function in various cellular processes, including transcription, splicing, and translation. Dysregulation of RNA structural ensembles has been linked to human diseases, highlighting the importance of studying these structures. Emerging technologies are enabling the direct measurement of RNA structural ensembles in living cells, providing new insights into their roles in RNA function and disease. The ability of RNAs to adopt multiple structures allows them to adapt to different cellular contexts and encode specific functional outcomes. Therapeutic targeting of alternative RNA states is a promising approach for modulating RNA activity. The study of RNA structural ensembles is a rapidly evolving field with significant implications for both basic research and clinical applications.RNA secondary structure ensembles and alternative states play critical roles in RNA function and regulation. Most RNAs can fold into multiple alternative structures, and these ensembles are dynamically regulated by cellular processes. Understanding these ensembles is essential for elucidating RNA biology and developing therapeutic strategies. RNA structural ensembles are influenced by factors such as RNA sequence, post-transcriptional modifications, and interactions with proteins and ions. These ensembles are also critical for RNA function in various cellular processes, including transcription, splicing, and translation. Dysregulation of RNA structural ensembles has been linked to human diseases, highlighting the importance of studying these structures. Emerging technologies are enabling the direct measurement of RNA structural ensembles in living cells, providing new insights into their roles in RNA function and disease. The ability of RNAs to adopt multiple structures allows them to adapt to different cellular contexts and encode specific functional outcomes. Therapeutic targeting of alternative RNA states is a promising approach for modulating RNA activity. The study of RNA structural ensembles is a rapidly evolving field with significant implications for both basic research and clinical applications.