Building Synthetic Long Non-Coding RNAs via Iterative Golden Gate Assembly of K-mer Arrays v1
Year: 2025
Type: preprint
Abstract: Long noncoding RNAs (lncRNAs), a class of noncoding RNAs exceeding 500 nucleotides and transcribed mostly by RNA polymerase II, regulate gene transcription and chromatin organization. Acting as molecular guides, scaffolds, and structural components, lncRNAs interact with RNAs, DNA, and proteins. Repeat motifs within lncRNAs called k-mers are associated with protein interactions and chromatin complex recruitment. Understanding lncRNA mechanisms is hampered by tissue specificity, redundancy, and multifunctionality, necessitating quantitative investigation. To support the systematic investigation of repeat motifs, we optimized Golden Gate assembly with standardized 4-nucleotide linkers to assemble any four lncRNA elements into 4x k-mers without the need to design compatible overhangs. To extend the length (repeat number) of RNA constructs, a 2x BbsI Golden Gate cloning site is restored at the 3’ end of the assembled 4x k-mer, allowing the addition of more k-mers to generate an 8x k-mer, 12x k-mer, and so on. Please read the Guidelines for suggestions on how to make arrays of intermediate lengths (e.g. 1x, 2x, 3x, 5x, 6x, 7x, etc.). Beginning with k-mer identification, synthetic lncRNAs can be constructed and expressed in vitro or in cells within three weeks, offering an efficient framework to study and harness their regulatory functions.
Authors: Seong Hu Kim, Chloe Guerrero, Karmella A Haynes
Institution: Emory University
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
Funder: National Science Foundation
OpenAlex ID: W4406789343