Team:Uppsala University/Modelling

The modelling of our constructed sRNA is a vital part for evaluating hypotheses of how sRNA molecules interact with their target. One canonical approach for sRNA stability is to evaluate secondary structures by minimum free energy (MFE) approaches. This gives a pointer for approximating the probability of different kind of interactions.

Using a native scaffold to stabilize the interactions
The wildtype small RNA of E.coli K-12 MG1655 that we used as a template for constructing our own smallRNA is named Spot42 and has been shown to be interacting with the Hfq protein.

Spot42 has like many other smallRNA, two distinctive parts. One that binds to a mRNA sequence, and another sequence that interacts with the Hfq-rna binding protein that is belived to play an important role in the function of smallRNA. (Holmquist, 2012)

Here you can see the native spot42 with the scaffold marked in blue.

Picture of native spot42
To gain better understanding of the mechanisms of smallRNA translation inhibition we have made 2D models of all our found smallRNAs. The 2D models were calculated in CLC main workbench.
IntaRNA, an RNA-interaction prediction software adapted for sRNA and ncRNA interactions (Smith et. al., 2010) was used to predict the sRNA-mRNA interactions of the candidate sRNAs.

The sRNA were isolated, sequenced and analyzed to find the hybridizing base pairs. The sequencing of the sRNAs that showed downregulation of YFP also had a matching sequence in the 5’UTR of our target mRNA.
Altough, a few of the sRNA that downregulated YFP was shown to hybridize at the YFP mRNA region. Two of these were further studied and modelled. At last, a prediction of the structure between the sRNA UU17 and AAC(6’)UTR mRNA were modelled.

Below are two examples of structures that were found to match regions in the coding sequence of the SYFP2. It is apparent that false positives will appear when using a reporter system that itself can be repressed, but by modelling interactions with the target mRNA, we save much time knowing which sRNAs are not of direct interest.

dG = -28.3kcal/mol	Length in base pairs =82 Number of hybridizing base pairs =10 Maximum number of hybridizing bases in a row = 6 Gibbs free energy = -5.6 kcal/mol

dG = -34.5 kcal/mol	Length in base pairs =85 Number of hybridizing base pairs =21 Maximum number of hybridizing bases in a row =9 Gibbs free energy = -10.9kcal/mol