The potential of synthetic mRNA devices to serve as gene therapies and vaccines are constrained by their short half-lives in living organisms. While it was originally believed that poly(A) tails composed of a long stretch of adenosines at the 3’ end of an mRNA, current mRNA sequencing technologies have unveiled the presence of non-adenosine nucleotides on natural poly(A) tail, which play regulatory roles in mRNA biology. This study aims to look into the natural poly(A) tail modifications that may affect the mRNA translation efficiency. Previous results have suggested that the location of non-A substitution in the terminal of an mRNA could affect the protein expression in cells. However, due to the imperfect synthetic mRNA synthesis method, we cannot evaluate the effect of non-A modifica...[ Read more ]

The potential of synthetic mRNA devices to serve as gene therapies and vaccines are constrained by their short half-lives in living organisms. While it was originally believed that poly(A) tails composed of a long stretch of adenosines at the 3’ end of an mRNA, current mRNA sequencing technologies have unveiled the presence of non-adenosine nucleotides on natural poly(A) tail, which play regulatory roles in mRNA biology. This study aims to look into the natural poly(A) tail modifications that may affect the mRNA translation efficiency. Previous results have suggested that the location of non-A substitution in the terminal of an mRNA could affect the protein expression in cells. However, due to the imperfect synthetic mRNA synthesis method, we cannot evaluate the effect of non-A modifications in specific locations within the tail. In this project, we successfully demonstrated that shifting a single cytidine or guanine by one position of the poly(A) tail can alter protein production. Next, we extracted the natural non-A fragments existing on HeLa S3 mRNAs and tested their effect on synthetic mRNA translation efficiency. One fragment, GGCCCCU, exhibited a significant effect on impeding mRNA stability and translation capability by triggering a strong innate immune response. In the future, we intend to investigate how the position of cytidine and guanine can affect poly(A) tail degradation pathway that potentially affect the protein expression. Moreover, we will test if the fragment, GGCCCCU could also reduce mRNA translation efficiency when using a transfection method that avoid triggering innate immune response. By identifying non-canonical tails that can affect protein expression, we hope that this study will facilitate the optimization of poly(A) tails for the design of synthetic mRNA and contribute to the development of mRNA therapeutics.