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

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Virology and Drug Delivery

Background:

• Specific RNA encapsulation in cells is a complex molecular recognition challenge.
• Engineered protein capsids, like the positively supercharged Aquifex aeolicus lumazine synthase (AaLS-pos), can encapsulate cellular RNAs.
• Previous work demonstrated charge complementarity drives encapsulation in AaLS-pos.

Purpose of the Study:

• To determine the impact of nucleotide sequence on RNA encapsulation efficiency.
• To investigate how RNA sequence influences partitioning between capsids and the cellular environment.
• To explore factors governing guest selection in AaLS-pos systems.

Main Methods:

• Co-production of engineered AaLS-pos capsids with eight reporter RNAs in Escherichia coli.
• Reporter RNAs shared identical charge but possessed diverse arbitrary sequence regions (ASRs).
• Quantification of encapsulated full-length RNA using quantitative reverse transcription PCR (q-RT-PCR) and fluorescence assays.

Main Results:

• Significant variations in encapsulated RNA yields (up to 200-fold) were observed, driven by ASRs.
• The presence of the Broccoli-F30 aptamer significantly hindered encapsulation.
• RNA partitioning varied up to 28,000-fold, with Broccoli-F30-lacking RNAs showing higher capsid enrichment.

Conclusions:

• RNA encapsulation efficiency is strongly influenced by nucleotide sequence, beyond overall charge.
• RNA flexibility and shape complementarity with the capsid are critical factors in guest selection.
• Encapsulation efficiency inversely correlated with expression levels and predicted secondary structure stability for some reporters.