Watt, A. T. | bioRxiv Science

bioRxiv2026

Tuning siRNA Specificity through Seed Region Incorporation of Deoxyribonucleotide Stereoisomers

Precise chemical design continues to drive advances in RNA-based therapeutics. Here, we report the synthesis and site-specific incorporation of four canonical phosphoramidites (U, C, A, and G), each bearing non-natural nucleoside configurations: {beta}-D-2'-deoxyxylonucleosides and -L-2'-deoxyribonucleosides. These stereochemically distinct nucleoside analogs were introduced at positions 6 and 7 within siRNA seed regions. When applied to siRNAs targeting Ttr, ACTN1, and Marc1, these modifications reduced off-target gene repression in functional assays and, in several cases, in transcriptome-wide differential expression analyses, while preserving robust on-target activity. In vivo, Marc1-targeting siRNAs containing these modified nucleosides showed decreased hepatotoxicity, as evidenced by reduced serum ALT and AST levels. Collectively, these findings establish {beta}-D-2'-deoxyxylonucleoside and -L-2'-deoxyribonucleoside analogs as promising chemical tools for enhancing the specificity and safety of siRNA therapeutics. This work underscores the power of integrating rational nucleoside design with comprehensive functional and in vivo evaluation to advance drug development based on RNA interference (RNAi). GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/699368v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@10e6e8aorg.highwire.dtl.DTLVardef@7b2797org.highwire.dtl.DTLVardef@1643f44org.highwire.dtl.DTLVardef@75a0a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Mistimed feeding disrupts circadian rhythms of male mating behavior and female preovulatory LH surges in mice

In rodents, eating at atypical circadian times, such as during the biological rest phase when feeding is normally minimal, reduces fertility. Prior findings suggest this fertility impairment is due, at least in part, to reduced mating success. However, the physiological and behavioral mechanisms underlying this reproductive suppression are not known. In the present study, we tested the hypothesis that mistimed feeding-induced infertility is due to a disruption in the normal circadian timing of mating behavior and/or the generation of pre-ovulatory luteinizing hormone (LH) surges (estrogen positive feedback). In the first experiment, male+female mouse pairs, acclimated to be food restricted to either the light (mistimed feeding) or dark (control feeding) phase, were scored for mounting frequency and ejaculations over 96 hours. Male mounting behavior and ejaculations were distributed much more widely across the day in light-fed mice than in dark-fed controls and fewer light-fed males ejaculated. In the second experiment, the timing of the LH surge, a well characterized circadian event driven by estradiol (E2) and the SCN, was analyzed from serial blood samples taken from ovariectomized and E2-primed female mice that were light-, dark-, or ad-lib-fed. LH concentrations peaked 2h after lights-off in both dark-fed and ad-lib control females, as expected, but not in light-fed females. Instead, the normally clustered LH surges were distributed widely with high inter-mouse variability in the light-fed group. These data indicate that mistimed feeding disrupts the temporal control of the neural processes underlying both ovulation and mating behavior, contributing to subfertility.

physiology↗