Guest Sequence Can Influence RNA Encapsulation by an Engineered Cationic Protein Capsid.

Abstract

Specific encapsulation of RNA in a cell is a challenging molecular recognition problem that has important implications for virology and drug delivery. An engineered variant of the Aquifex aeolicus lumazine synthase capsid that possesses a positively supercharged interior (AaLS-pos) has previously been shown to encapsulate a mixture of cellular RNAs in bacteria via charge complementarity. To investigate the influence of nucleotide sequence on encapsulation, eight reporter RNAs with the same charge but with highly diverse arbitrary sequence regions (ASRs) were coproduced with AaLS-pos in Escherichia coli cells. The ASRs cause significant differences in the yields of encapsulated full-length reporter RNA, measured by q-RT-PCR. Four of the reporters also have the Broccoli-F30 aptamer, which hinders encapsulation compared with reporters with an alternative sequence in place of Broccoli-F30. In all, the encapsulation yield of the best reporter was ∼200 times higher than the worst. The reporters also differed by up to ∼28 000 times in their partitioning between the capsid and the bulk cellular environment, with those lacking Broccoli-F30 showing higher enrichments in AaLS-pos. The detection of Broccoli-F30 by a fluorescence assay showed higher levels than that determined by q-RT-PCR, suggesting that partially degraded forms of the reporters are encapsulated more frequently than the full-length versions. For the reporters lacking the Broccoli-F30 aptamer, the encapsulation yield shows inverse correlations with both their expression levels and the predicted secondary structural stabilities of their ASRs. These observations suggest that in this system, guest selection depends on the RNA flexibility and the shape complementarity with the capsid.