The pRNA-assisted RNA imaging scheme (pARIS): a generalizable scaffold-based approach to facilitate cryo-EM structure determination of small RNAs

Seth Scott; Nikolai Prokhorov; Wei Zhao; Paul J. Jardine; Marc C. Morais; Kyung H. Choi; Seth Scott; Nikolai Prokhorov; Wei Zhao; Paul J. Jardine; Marc C. Morais; Kyung H. Choi

doi:10.3934/biophy.2026017

AIMS Biophysics

2026, Volume 13, Issue 3: 287-301. doi: 10.3934/biophy.2026017

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The pRNA-assisted RNA imaging scheme (pARIS): a generalizable scaffold-based approach to facilitate cryo-EM structure determination of small RNAs

1.
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology, The University of Texas Medical Branch, Galveston, TX 77555, USA
2.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
3.
Department of Diagnostic and Biological Sciences, School of Dentistry, and Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA

Received: 15 May 2026 Revised: 07 July 2026 Accepted: 15 July 2026 Published: 31 July 2026

RNA performs diverse biological functions that are often encoded in its three-dimensional structure. However, RNA-only structures represent a small fraction (~1%) of entries in the Protein Data Bank (wwPDB), limiting our knowledge of RNA structure–function relationships. The ribose-phosphate backbone of RNA is relatively flexible compared to proteins and typically forms weaker long-range tertiary interactions, resulting in structures that are less amenable to X-ray crystallography than folded proteins. Further, many functional RNAs range from 50 to 200 nt in size and are often too small for structure determination by cryo electron microscopy (cryo-EM). To facilitate RNA structure determination by cryo-EM, we present the procapsid RNA (pRNA)-assisted RNA imaging scheme (pARIS). In this approach, the RNA of interest is fused to the pRNA of bacteriophage Φ29 to enhance stability and reduce sample heterogeneity. The pRNA-linked RNA is then assembled onto the Φ29 procapsid to form a stable pentameric complex. This approach increases effective molecular mass, improves signal-to-noise ratio, and facilitates particle picking and alignments during cryo-EM imaging processing. Using pARIS, we determined a 4.8 Å structure of a 70 nt (~23 kDa) tRNA as proof of principle and a 6.7 Å structure of the liver-specific host microRNA-122 bound to its target site in the hepatitis C virus genome, demonstrating applicability to biologically relevant RNA targets.
- cryogenic electron microscopy,
- cryo-EM,
- RNA structure,
- RNA scaffold,
- bacteriophage Φ29,
- pRNA,
- pRNA scaffold,
- tRNA,
- HCV 5′,
- UTR,
- miR-122
Citation: Seth Scott, Nikolai Prokhorov, Wei Zhao, Paul J. Jardine, Marc C. Morais, Kyung H. Choi. The pRNA-assisted RNA imaging scheme (pARIS): a generalizable scaffold-based approach to facilitate cryo-EM structure determination of small RNAs[J]. AIMS Biophysics, 2026, 13(3): 287-301. doi: 10.3934/biophy.2026017

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Abstract

RNA performs diverse biological functions that are often encoded in its three-dimensional structure. However, RNA-only structures represent a small fraction (~1%) of entries in the Protein Data Bank (wwPDB), limiting our knowledge of RNA structure–function relationships. The ribose-phosphate backbone of RNA is relatively flexible compared to proteins and typically forms weaker long-range tertiary interactions, resulting in structures that are less amenable to X-ray crystallography than folded proteins. Further, many functional RNAs range from 50 to 200 nt in size and are often too small for structure determination by cryo electron microscopy (cryo-EM). To facilitate RNA structure determination by cryo-EM, we present the procapsid RNA (pRNA)-assisted RNA imaging scheme (pARIS). In this approach, the RNA of interest is fused to the pRNA of bacteriophage Φ29 to enhance stability and reduce sample heterogeneity. The pRNA-linked RNA is then assembled onto the Φ29 procapsid to form a stable pentameric complex. This approach increases effective molecular mass, improves signal-to-noise ratio, and facilitates particle picking and alignments during cryo-EM imaging processing. Using pARIS, we determined a 4.8 Å structure of a 70 nt (~23 kDa) tRNA as proof of principle and a 6.7 Å structure of the liver-specific host microRNA-122 bound to its target site in the hepatitis C virus genome, demonstrating applicability to biologically relevant RNA targets.

Acknowledgments

This work was supported by NIH grants R01AI182321 (to MCM and PJJ), R01GM122979 (to MCM and PJJ), R01GM127365 (to MCM), R01AI087856 (to KHC), R21AI181621 (to KHC), and T32GM008280 (to SS). We would also like to acknowledge the Sealy Center for Structural Biology and Molecular Biophysics (SCSB) for support of the UTMB cryo-EM and computational core facilities.

Conflict of interest

All authors declare no conflicts of interest in this paper.

Author contributions

M.C.M. and K.H.C. conceived the study. S.S. processed the pRNA-HCV: miR-122 cryo-EM data, contributed to figure preparation, and assisted with manuscript writing. N.P. collected and processed the pRNA-tRNA cryo-EM data. K.H.C. also contributed to processing of the pRNA–tRNA data. W.Z. and P.J.J. contributed procapsid and pRNA reagents, and P.J.J. contributed to the conceptualization of the circularly permuted RNA strategy. M.C.M. and K.H.C. wrote and edited the manuscript with input from all authors. All authors read and approved the final manuscript.

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