Optimizing splinted ligation of highly structured small RNAs

Abstract

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73 mer tRNA, the other a 49 mer synthetic ribozyme. A stable LCC could be isolated and turned over with>95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

FIGURE 1.

Splint ligation of mitochondrial tRNA^Lys. (A) composition of ARI, DRI, and LCC; phosphates necessary for ligation are indicated by “p,” and hydroxyl groups necessary for ligation are indicated by “OH”.; DNA splints are in light gray; the phosphate acceptor RNA is in green and the donor RNA in red throughout the figure. (B) Splints of 30, 40, 50, and 73 nucleotides and RNA fragments TR5 and TR3. (C) tRNA^Lys with attachment sites for Cy3 and Cy5. The ligation site is indicated in blue. (D) PAGE of ligation reactions mediated by a 30mer splint at different temperatures. (Lane 1) Unligated fragments TR3,TR5. (Lanes 2–5) Ligation reactions at 15° C, 20° C, 25° C, and 30° C. (Lane 6) Contains size markers, TR3, TR5, and full-length product originating from an unrelated synthesis. Total ligation yield and full-length product yield are indicated below the PAGE. (E) Dependence of ligation yield on splint DNA length. (Lane 1) Standard ligation without cDNA template. (Lane 2) Standard ligation to which T4-DNA ligase was added after incubation with DNase. (Lane 3) Unligated fragments TR3, TR5. (Lanes 4–7) Standard ligations with splints of different length. Samples in lanes 5,7,9,11 were incubated with DNase after the ligation reaction. (F) SEC profiles of hybridization mixtures monitored by absorption at different wavelengths. The length of the splint is indicated in each panel. ARIs, DRIs, RNA dimers, LCCs, and structural isoforms as identified by accompanying titration experiments are indicated.

FIGURE 2.

Splint ligation of a 49mer ribozyme. Three splint DNAs of 30, 40, and 49 nucleotides (nt) and the RNA fragments RZ5 (green) and RZ3 (red) were used in ligation studies. The color code is maintained throughout the figure. (A) Target ribozyme with attachment sites for Cy3 and Cy5. The ligation site is indicated in blue. (B) Dependence of ligation yield on splint DNA length. (Lane 1) Standard ligation without cDNA template. (Lane 2) Standard ligation to which T4-DNA ligase was added after incubation with DNase. (Lane 3) Unligated fragments RZ3, RZ5. (Lanes 4–6) Standard ligations with splint oligos of 49, 40, and 30 nt in length. As indicated by white lines between lanes 4 and 6, these lanes stem from the same gel, but lanes containing nonrelevant experiments have been omitted. (C) SEC profiles of hybridization mixtures monitored by absorption at different wavelengths and by FRET detection. The length of the splint is indicated in each panel. ARIs, DRIs, RNA dimers, LCCs, and structural isoforms are indicated.