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Transfer RNAs (tRNAs) purified from non-pathogenic E. coli strain (NPECS) possess cytotoxic properties on colorectal cancer cells. In the present study, the bioactivity of tRNA halves and tRNA fragments (tRFs) derived from NPECS are investigated for their anticancer potential. Both tRNA halves and tRF mimics studied exhibited significant cytotoxicity on colorectal cancer cells, with the latter being more effective suggesting that tRFs may be important contributors to the bioactivities of tRNAs derived from gut microbiota. Through high-throughput screening, EC83 mimic, a double-strand RNA with a 22 nt 5’-tRF derived from tRNA-Leu(CAA) as antisense chain, was identified as one with the highest potency (IC50=52 nM). Structure-activity investigations revealed that 2’-O-methylation of the ribose of guanosine may enhance the cytotoxic effects of EC83 mimic via increasing the stability of its tertiary structure. Consistently, 4-thiouridine substitution reversed this increased stability and the enhanced cytotoxic effects. This provides the first evidence that the bioactivity of tRF mimics would be impacted by chemical modifications. Furthermore, the present study provides the first evidence to suggest that novel tRNA fragments derived from the gut microbiota may possess anti-cancer properties and have the potential to be potent and selective therapeutic molecules.IMPORTANCEWhile the gut microbiota has been increasingly recognized to be of vital importance to human health and disease, the current literature shows that there is a lack of attention given on the non-pathogenic Escherichia coli strain. Moreover, the biological activities of tRNA fragments (tRFs) derived from bacteria have rarely been investigated. The findings from this study revealed tRFs as a new class of bioactive constituents derived from gut microorganisms, suggesting that studies on biological functional molecules in intestinal microbiota should not neglect tRFs. The research of tRFs would play an important role in biological research of gut microorganisms, including bacteria-bacteria interaction, gut-brain axis, gut-liver axis, etc. Furthermore, the guidance on the rational design of tRF therapeutics provided in this study indicates that further investigations should pay more attention to these therapeutics from probiotics. The innovative drug research of tRFs as potent druggable RNA molecules derived from intestinal microorganisms would open a new area in biomedical sciences.