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Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription. [Copyright &y& Elsevier]

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc; and a founder, Scientific Advisory Board (SAB) member of, and consultant to ReOpen Diagnostics, LLC. J.D.B. also serves or served on the SABs of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. Y.A. is a Founder and CSO of Logomix, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)