Differences of tsRNA expression profiles efficiently discriminate monozygotic twins in peripheral blood.

Monozygotic twins (MZTs) share nearly identical genomic DNA sequences, making traditional forensic short tandem repeats (STR) genotyping methods ineffective for distinguishing between them. In recent years, the use of epigenetic factors in forensic applications has gained traction. The dynamic epigenetic factors can be influenced by inherited traits or acquired environmental factors. This study analyzed the expression profiles of transfer RNA-derived small RNAs (tsRNAs) in peripheral blood from four pairs of adult MZTs using Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing (PANDORA-seq). Differentially expressed tsRNAs (DEtsRNAs) were identified and validated using the reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (ddPCR) in both adult and newborn MZTs. The study also evaluated the longitudinal temporal stability, resistance to degradation, and suitability of DEtsRNAs for aged bloodstains. A total of 8795 expressed tsRNAs were identified in the four pairs of adult MZTs by PANDORA-seq. After screening with a normalized | log (fold change) | > 1 and an adjusted p-value < 0.05, 10, 187, and 1520 DEtsRNAs were shared by 4, 3, and 2 pairs of MZTs. RT-qPCR and ddPCR confirmed the expression of the 10 DEtsRNAs identified by PANDORA-seq. Six candidate tsRNAs (tRNA-Gly-GCC, tRNA-Leu-TAA, tRNA-Lys-CTT, tRNA-Val-AAC_5_end, tRNA-iMet-CAT_5_end, and tsRNA-3023a/b-PheGAA) were identified as effective discrimination markers, even in neonatal MZTs which are largely unaffected by environment factors. Forensic applicability assessment revealed that tRNA-Gly-GCC and tRNA-Leu-TAA remained detectable in the 180-day-series bloodstains, while tRNA-Lys-CTT, tRNA-Val-AAC_5_end, and tRNA-iMet-CAT_5_end were relatively stable after 15 times of freeze-thaw cycles. Additionally, tRNA-Gly-GCC and tRNA-Lys-CTT exhibited long-term stability, with consistent expression over six months. In conclusion, this study demonstrates that differential tsRNAs expression can serve as a novel biomarker for MZT identification in forensic medicine.