Ligation of puromycin containing DNA linker to the 3’ end of mRNA using Moore-Sharp splinted ligation is a crucial step in preparation of fusible mRNA template. The efficiency of this ligation reaction, however, depends notably on the sequences of the mRNA. When long mRNA is used, the yield of ligated product is so low that large-scale transcription and ligation should be performed in order to get enough templates for translation. Compared to splinted ligation, psoralen mediated crosslinking is much more efficient in preparation of fusible long RNA templates.

Psoralens are tricyclic furocumarins with extended aromaticity, which have a strong tendency to intercalate with DNA base pairs. Irradiation of nucleic acids in the presence of psoralen with long wave UV (~360 nm) results in the 2+2 cycloaddition of either of its two photoreactive sites with 5,6-carbon bonds of pyrimidines. This feature makes psoralen very useful in crosslinking double-stranded nucleic acids. Usually this is realized by targeting the desired RNA region using a short complementary DNA containing a psoralen at its 5’ terminus.

The interaction between short DNA and mRNA is usually not strong, especially when the mRNA is structured. To stabilize interactions between mRNA and short DNA linker, I used a DNA linker containing a few 2’-O-methyl nucleotides after the 5’ psoralen residue. 2’-O-methyl oligos have advantages such as RNAse H resistance, much higher affinities for RNA, faster kinetics of hybridization, and ability to bind structured targets. Also, short 2’-O-methyl oligos can discriminate between matched and mismatched RNA targets much better than longer normal DNAs. Such oligos turned out to be very effective in crosslinking long RNA templates used for fusion reaction.

3’ end of mRNA: …………CACCGGCUA

DNA linker:

5’- Pso (TAGCCGGTG)_m(AAAAAAAAAAAAAAAAACC)_dPu

Pso: C2- or C6-Psoralen

( )_m: 2’-O-methyl RNA

( )_d: standard DNA

Crosslinking Protocol:

20 mM HEPES, pH7.4

100 mM KCl

1 mM spermidine

5 mM mRNA

10 mM Pso-DNA linker

Heat at 70°C for 5 mins, cool to 25°C in 5 mins

Transfer to 96-well plate (100 ml each well)

Irradiate at 4°C with 366nm UV lamp for 15 to 20 mins

Load to high capacity quick-spin G-50 (optional)

Use for translation/fusion

Note:

1. Crosslinking yield is approximately 40-60% after 15 mins. Long time irradiation does not give more products.
2. Flexible spacers such as C9 may be used. However, the total length of DNA linker should be less than 35 nucleotides.
3. If 2’-O-methyl nucleotides are not used, C6-Psoralen gives better crosslinking result.
4. Avoid using 254 nm UV since the monoadducts and crosslinks can be photoreversed by irradiation with UV at that wavelength.