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.