Stabilization of RNA hairpins using non-nucleotide linkers and circularization

Abstract

An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies.

Figure 1.

Stabilization of the hairpin structure by non-nucleotide linkers (-L-) following enzymatic circularization. Structures of the stilbene diether and hexaethylene glycol (EG6) linkers are depicted.

Figure 2.

Circularization of RNA using T4 RNA ligase. (A) Schematic representation of RNA substrates: three oligomers containing CUG repeats and a sarcin–ricin (SR–G) sequence from Escherichia coli 23S rRNA. (B) Control (C) and ligation reactions (L) resolved on an 8% polyacrylamide gel in denaturing conditions. Open and circular forms of the RNA oligomers are indicated.

Figure 3.

Identification of the circular form of the RNA oligomer. (A) Circularization reaction was carried out in the absence of ATP or T4 RNA ligase and separated on an 8% polyacrylamide gel. C—RNA oligomer only. (B) Autoradiogram shows separation of the products of alkaline hydrolysis to the linear and circular forms of the RNA oligomer (R). C—control reaction. (C) Identification of the circular RNA form using dephosphorylation. The reaction products were separated on an 8% polyacrylamide gel and visualized by autoradiography followed by staining with SYBR Green II.

Figure 4.

(A) Circularization efficiency of RNA using recombinant (rec.) or commercially available enzymes (lanes A and B). The ligation efficiency is not affected by the annealing temperature (B) but depends on the ligation temperature. (C) Open and circular forms of the RNA oligomer are indicated.

Figure 5.

The normalized UV melting curves of the open (orange) and circular (blue) form of the 8G RNA oligomer.

Figure 6.

Stabilization of the hairpin structure by the click reaction. Red and blue triangles represent the 5΄ and 3΄ end modifications required for circularization using the click reaction. The triazole linkage is depicted.

Figure 7.

Circularization of DNA and RNA oligomers using the click chemistry. (A) Separation of the click reaction products (R) along with the linear form of the oligomer (C). (B) Schematic representation of the open and closed forms of DNA oligomer. The HpaII restriction sites and length of the digestion products are marked. (C) 20% polyacrylamide gel electrophoresis (PAGE) of the restriction products of open and circular forms of the DNA oligomer. C—control lane.