Substrate design

The selection of a suitable substrate has a large influence on the data obtainable by CPA. To achieve as much information as possible, it is important to use a substrate with certain properties. The substrate should consist of at least two possible cleavage positions and the possible cleavage products should differ in their mass to be distinguished by MS. Further on, there should be no intramolecular interactions forming a secondary structure which interferes with the enzymatic reaction.

To investigate, whether RNase T1 distinguishs between a cleavage sequence near the 5' terminus and the 3' terminus, a substrate had to be constructed with two cleavage sequences, each near the respective terminus. To exclude all other influences, to identical cleavage sequences were linked together to form a substrate with two definite cleavage sequences, each near the 5' respectively the 3' terminus. As described in 1.2.2, it can be assumed that the optimal short substrate for RNase T1 has the sequence 5'-pApGpC-3' . Therefore the simplest substrate with the properties desired would have the following sequence: 5'-pApGpCpApGpC-3' . However, introducing an uracil at the 5' terminus of the original sequence has two advantages: first, the distance between both cleavage sequences is enhanced, and, second, it is an easy way to introduce the phosphate group at the 5' terminus of the sequence. Hence, the substrate used (R/8) had the sequence

and has two different cleavage positions (see figure 3.15) which can lead to eight different cleavage products for RNase T1 as shown in figure 3.16.

Figure 3.15: Possible cleavage positions of R/8 for an enzyme with adenosine or guanosine specificity. $\alpha$ and $\beta$ indicate the point of attack for an enzyme with guanosine specificity, $\gamma$ and $\delta$ for an enzyme with adenosine specificity.

Figure 3.16: R/8 and its possible RNase T1 hydrolysis products with molecular mass (average isotope mass, [M$+$H$^+$]$^+$) and possible intermediates (the first mass represents the molecular weight of the 2',3' cyclic phosphodiester, the second one the molecular weight of the final product, the 3' phosphate.)

Figure 3.17: Mass spectrum of the CPA substrate R/8. Next to the [M+H$^+$]$^+$ of R/8, sodium and potassium adducts can be found.
Conditions: 100 $\mu$M R/8, 3-hydroxypicolinic acid