RNase 4 (NEB #M1284) DNA Probe-Directed Analysis of mRNA 5´Cap Structures

Optimized for 5 μg of a 2000 nucleotide mRNA-of-interest

1. In a 0.2 ml PCR tube combine 5 μg of mRNA with 40 pmol of a biotinylated DNA probe in 10 mM Tris pH 7.0 to a final 10 µl volume. (Note #1)
   
   
2. To hybridize, heat the RNA:DNA mixture in a thermal cycler to 80°C followed by 0.1℃/s temperature ramp down to 22℃.
   
   
3. In a 1.5 mL centrifuge tube dilute the hybrid RNA:DNA duplex into 20 μL of 1.5 X NEBuffer™ r1.1, for a final 30 µl volume.
   
   
4. Add 1 μL of a 1:30 dilution of RNase 4. Dilution should be performed in 1 X NEBuffer™ r1.1 (Note #2).
   
   
5. Incubate the digest for 1 h at 37°C.
   
   
6. Stop the RNase 4 digest with 1 μL of RNase inhibitor, Murine (NEB #M0314) followed by incubation at room temperature for 10 minutes. (Note #3)
   
   
7. Proceed to isolation of hybridized RNA:DNA duplexes using streptavidin magnetic beads (NEB #S1421) and downstream LC-MS/MS analysis. (Note #4)

Note #1: Protocol designed with ~2-fold molar excess of reverse-complement DNA probe to target RNA 5′ ends. DNA probes at least 20 nt in length have been tested. Probes can be designed with either 3´ or 5´ biotin/desthiobiotin affinity groups for streptavidin enrichment (NEB #S1421). For optimal results, the protected DNA:RNA hybrid region should be 4 or 5 nucleotides upstream of the target RNase 4 U/A or U/G cut site.

Note #2: It is recommended to dilute RNase 4 in 1 X NEB r1.1. A 10-fold to 50-fold dilution range of RNase 4 (NEB #M1284) is optimal for analysis of 5 µg of a 2000 nucleotide mRNA sequence. Unused, diluted RNase 4 should be discarded. To obtain mixtures of well-defined 5´ end products for other lengths and amounts RNA, optimal dilutions of RNase 4 need to be empirically determined.

Note #3: Both RNase inhibitor, Human Placenta (NEB #M0307) or Murine (NEB #M0314) inhibit RNase 4 activity.  

Note #4: RNase 4 product fragments will contain a mixture of linear 3´-P and cyclic 2´, 3´ P termini.