Modular RNA:DNA Nanostructures Enable Nanopore Profiling of rRNA Processing and rRNA Variants

Area of Science:

• Molecular Biology
• Nanotechnology
• Genomics

Background:

• Ribosomal RNAs (rRNAs) are crucial species-defining markers but are difficult to analyze directly due to enzyme biases and high sequence conservation.
• Distinguishing closely related rRNA variants and studying rRNA processing, like intervening sequence (IVS) excision, presents significant challenges in molecular diagnostics.

Purpose of the Study:

• To develop a method for direct identification and analysis of native rRNAs and their variants without enzyme-induced biases.
• To enable the resolution of rRNA processing states and single-nucleotide differences in rRNA sequences.

Main Methods:

• Development of modular RNA:DNA nanostructures (RNA identifiers or IDs) assembled onto native rRNAs.
• Utilized short complementary oligonucleotides with programmable coding motifs for RNA ID assembly.
• Detection of RNA IDs using solid-state nanopores and incorporation of catalytically inactive Cas9 for single-nucleotide discrimination.

Main Results:

• Demonstrated direct conversion and detection of native bacterial 16S rRNAs into RNA IDs using nanopore technology.
• Successfully resolved biologically encoded rRNA processing states, including serovar-specific 23S rRNA fragmentation patterns from IVS excision.
• Achieved single-nucleotide discrimination of rRNA variants by integrating Cas9 ribonucleoprotein complexes.

Conclusions:

• The modular RNA ID nanopore system provides a direct readout of native rRNAs, overcoming limitations of traditional methods.
• This approach facilitates the study of complex rRNA processing pathways and the characterization of diverse rRNA variants.
• The technology holds promise for advancing research in microbial identification, population genetics, and molecular diagnostics.