I am not an expert in this field, but I am very interested and have researched to find an answer. Could you please review the response below to see if it is correct?

To create a site-specific mutation in an lncRNA for a luciferase assay, you should first clone the entire lncRNA sequence into an expression vector using PCR. To mutate the miRNA interaction site within the lncRNA, employ site-directed mutagenesis. This technique utilizes specifically designed primers containing the desired mutation and a high-fidelity DNA polymerase to amplify the mutated sequence. Once the mutant construct is verified, both the wild-type and mutated lncRNA constructs can be co-transfected with the luciferase reporter vector into cells. The dual-luciferase reporter assay will then assess the interaction between the lncRNA and miRNA [1][2].

Reference

[1] Chen, W., Li, Q., Zhang, G., Wang, H., Zhu, Z., & Chen, L. (2020). LncRNA HOXA‐AS3 promotes the malignancy of glioblastoma through regulating miR‐455‐5p/USP3 axis. Journal of Cellular and Molecular Medicine, 24, 11755 - 11767.

[2] (Pu), H. E., Cai, J., Weissensee, P., Sharma, P., Tocci, M., & Wright, P. (2011). A Cell-Based Protein-Protein Interaction Method Using a Permuted Luciferase Reporter. Current Chemical Genomics, 5, 122 - 128.

I received some assistance from tlooto.com for this response.

I would transfer the lncRNA fragment of interest to the 3'UTR of your favorite reporter plasmid. You basically have 2 options, mutate or substitute with a synthetic fragment. For synthetic, I like HiFi assembly from New England Biolabs. Just buy a Wt and a mutated version of the region of interest (which should be pretty cheap since small) and follow their guidelines. If your region is fairly small (only the miRNA cognate sequence), I would PCR it in the reporter plasmid by insertion mutagenesis in your reporter construct using oligos containing the sequence of interest. Several ways to do that and New England Biolabs has a nice kit (which also works for site-directed and deletion approaches).

https://www.neb.com/en/products/e0554-q5-site-directed-mutagenesis-kit

Good luck!