Generating accurate alignments of non-coding RNA sequences is indispensable in the quest for understanding RNA function. Nevertheless, aligning RNAs remains a challenging computational task. In the twilight-zone of RNA sequences with low sequence similarity, sequence homologies and compatible, favorable (a priori unknown) structures can be inferred only in dependency of each other. Thus, simultaneous alignment and folding (SA&F) remains the gold-standard of comparative RNA analysis, even if this method is computationally highly demanding. This text introduces to the recent release 2.0 of the software package LocARNA, focusing on its practical application. The package enables versatile, fast and accurate analysis of multiple RNAs. For this purpose, it implements SA&F algorithms in a specific, lightweight flavor that makes them routinely applicable in large scale. Its high performance is achieved by combining ensemble-based sparsification of the structure space and banding strategies. Probabilistic banding strongly improves the performance of LocARNA 2.0 even over previous releases, while simplifying its effective use. Enabling flexible application to various use cases, LocARNA provides tools to globally and locally compare, cluster and multiply align RNAs based on optimization and probabilistic variants of SA&F, which optionally integrate prior knowledge, expressible by anchor and structure constraints.