Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs

Background

PolyU as a Physical Object: PolyU is an abiological RNA molecule composed of repeated uridine residues, and therefore, it cannot Watson-Crick base-pair and is devoid of RNA secondary structure (Martin and Ames, 1962; Richards et al., 1963). PolyU has very weak base-stacking energy resulting in a lack of helical ordering as well–except below 4 °C (Richards et al., 1963). Due to this lack of structure polyU RNA can be described physically as a random coil (Inners and Felsenfeld, 1970), as opposed to normal RNA molecules that form complicated secondary/tertiary structures, making them more compact physical objects (Yoffe et al., 2008; Fang et al., 2011; Gopal et al., 2012; Garmann et al., 2015).

We therefore expect polyU to be less compact than viral RNA of the same length. Indeed dynamic light scattering (DLS) measurements (see Figure 3) indicate that in RNA assembly buffer (see Recipes) at 25 °C, polyU molecules are about 25% larger in average diameter than branched RNAs of the same length. Additionally, we expect polyU to be more flexible than normal-composition RNAs, as it lacks secondary and tertiary structure, resulting in a more extended, flexible molecule.

Synthesis and Fractionation of Long PolyU RNAs: The synthesis protocol used herein was developed from existing protocols used by Vanzi et al. (2003) and van den Hout et al. (2011). Specifically, polyU RNAs were synthesized from Uridine diphosphate (UDP) monomers using the enzyme polynucleotide phosphorylase (PNPase), which indiscriminately adds nucleotide diphosphates (NDPs) to the 3' end of oligonucleotides, in this case a 20-nt-long polyU seed oligo with a 5' cy5 fluorescent label (Beren et al., 2017). It is important to note that the oligo sequence and length can be changed as desired, and it can be tagged at the 5' end with fluorophores, linkers, or modified bases, enabling the synthesis of polyU with customizable markers. Because of its lack of base-pairing, polyU cannot be visualized in electrophoresis gels by the usual intercalating nucleic acid stains. For this reason, we chose to utilize a fluorophore at the 5' end of the RNA. Additionally, it is possible to make polyA, polyG or polyC using the same PNPase, by simply adding ADP, GDP or CDP, respectively (Grunberg-Manago et al., 1956).

The synthesis reactions produce a polydisperse mixture of fluorescent polyU RNAs ranging in length from 200 to 10,000 nts, with shorter lengths being represented in significantly higher copy number as determined by fluorescence detection in denaturing agarose gel electrophoresis (see Figure 1). After running the RNA mixture alongside a denatured ssRNA ladder (see Figure 1), it was fractionated by electroeluting excised portions of the gel using the ladder as a reference. In fact, we often run many lanes of polyU in parallel to increase the amount of fractionated polyU produced from a given gel, as we usually recover between 30% and 60% of the RNA after gel electroelution.


Figure 1. Denaturing electrophoresis gel analysis of unfractionated polyU. Electrophoresis gel analysis of unfractionated polyU (red, cy5 fluorescence) after synthesis using PNPase, compared with a single-stranded (ss) RNA ladder (green, ethidium bromide nucleic acid stain). This gel illustrated the high amount of polydispersity in the polyU generated from the synthesis reaction. The brightest band in the RNA ladder is 3,000-nt-long ssRNA.

We used this process to fractionate polyU into the following length fractions: 500-1,500, 1,500-2,500, 2,500-3,500, 3,500-5,000, 5,000-7,000 and 7,000-9,000 nt. The amount of polyU purified was quantified using UV-Vis absorbance measurements, and the absorbance ratio at 260 nm/280 nm was used as a measure of RNA purity (A₂₆₀/A₂₈₀ greater than 2.0). These fractionated polyU samples were then examined on a denaturing agarose gel to demonstrate that single contiguous RNA bands were obtained (see Figure 2).


Figure 2. Denaturing electrophoresis gel analysis of fractionated polyU. From right to left: ssRNA ladder, 5,000-7,000, 4,000-5,000, 2,500-4,000, 1,500-2,500 and 500-1,500 nt polyU RNAs. The decrease in band intensity for higher-molecular-weight polyU molecules is due to the fact that an equal mass of polyU was loaded per lane, resulting in many fewer RNAs in the higher-molecular-weight fractions, and subsequently, a decreased fluorescence signal (because of there being only one label per RNA molecule). (Adapted from Beren et al., 2017)