A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair

Abstract

As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.

Hydrogen Bonding in Natural and Unnatural Base Pairs—A Local Vibrational Mode Study

“…Utilizing DNA as a material building block in molecular and structural engineering has already led to the creation of numerous molecular-assembly systems and materials at the nanoscale [ 19 ]. Substantial efforts have been made to expand the genetic alphabet of DNA by introducing other base pair combinations, so-called unnatural base pairs ( UBP ) to increase nucleic acid functionalities [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. Recently, modifications of DNA containing four NBP and four additional UBP which efficiently replicated [ 32 , 32 ] were reported.…”

Alternative Watson–Crick Synthetic Genetic Systems

“…For example, introduction of a single Z:P pair into the stem of an RNA riboswitch marginally increased the stability of that stem; a crystal structure showed essentially no geometric perturbation (Fig. 7, right) (Hernandez et al 2015). In DNA, the crystal structure of a single Z:P pair likewise shows no substantial deviation from Watson -Crick geometry (Zhang et al 2015) (Fig.…”

“…A 16-mer duplex with six consecutive Z:P pairs (center)(Georgiadis et al 2015). A single Z:P pair in an RNA riboswitch in four views A, B, C, and D each rotated 90˚(right)(Hernandez et al 2015).…”

Artificially Expanded Genetic Information Systems for New Aptamer Technologies

“…These biophysical studies were followed by crystallographic studies that showed that AEGIS components do, in fact, pair with Watson–Crick geometry. For example, the introduction of a single Z:P pair into the stem of a riboswitch marginally increased the stability of that stem; a crystal structure showed essentially no geometric perturbation [ 92 ] ( Figure 2 , right). In DNA, the crystal structure of a single Z:P pair likewise showed no substantial deviation from Watson–Crick geometry [ 93 ].…”

“… Crystal structures with Z:P AEGIS pairs: ( left ) A 16-mer duplex with six consecutive Z:P pairs [ 82 ]; ( right ) A single Z:P pair containing a RNA guanine riboswitch superimposed to its natural counterpart [ 92 ]. …”