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  1. Due to their intrinsic nuclease resistance, l -oligonucleotides are being increasingly utilized in the development of molecular tools and sensors. Yet, it remains challenging to synthesize long l -oligonucleotides, potential limiting future applications. Herein, we report straightforward and versitile approach to assemble long l -RNAs from two or more shorter fragments using T4 RNA ligase 1. We show that this approach is compatible with the assembly of several classes of functional l -RNA, which we highlight by generating a 124 nt l -RNA biosensor that functions in serum.