Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNAenzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for singlemolecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cationdependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one’s disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.