Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5‑Hydroxymethylcytosine

5-Hydroxymethylcytosine (hm5C) is an RNA modification
that has attracted significant interest because of the finding that
RNA hydroxymethylation can favor mRNA translation. For insight into
the mechanistic details of hm5C function to be obtained,
the availability of RNAs containing this modification at defined positions
that can be used for in vitro studies is highly desirable. In this
work, we present an eight-step route to 5-hydroxymethylcytidine (hm5rC) phosphoramidite for solid-phase synthesis of modified
RNA oligonucleotides. Furthermore, we examined the effects of hm5rC on RNA duplex stability and its impact on structure formation
using the sarcin-ricin loop (SRL) motif. Thermal denaturation experiments
revealed that hm5rC increases RNA duplex stability. By
contrast, when cytosine within an UNCG tetraloop motif was replaced
by hm5rC, the thermodynamic stability of the corresponding
hairpin fold was attenuated. Importantly, incorporation of hm5rC into the SRL motif resulted in an RNA crystal structure
at 0.85 Å resolution. Besides changes in the hydration pattern
at the site of modification, a slight opening of the hm5rC–G pair compared to the unmodified C–G in the native
structure was revealed.