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Peer-reviewedCC BY-NC-ND 4.0ACS Omega

The Dual-Ribozyme RNA Ladder and RNA-Calibrated ssDNA Marker System for Inexpensive RNA Size Standards

  • Yingying Zhao1,2
  • Jiaqi Lin1,3
  • Mengxiao Li1
  • Lin Huang1
  • Yuting Xie1
  • Jia Wang2
  1. 1Sun Yat-sen University , , ,
  2. 2Shenzhen Technology University , , ,
  3. 3Hubei University of Chinese Medicine , , ,
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Abstract

RNA size markers are essential for denaturing Urea-PAGE analysis, but commercial RNA ladders are expensive, available only in fixed size ranges, and require careful handling to prevent RNA degradation. Here, we developed a modular dual-ribozyme platform for preparing RNA size standards from single T7 transcription templates. Each construct contains a central RNA segment flanked by hammerhead and HDV ribozymes, enabling cotranscriptional self-cleavage to generate a predictable set of RNA products. The SYSU25 construct reproducibly produced short RNA marker bands at 25, 50, and 75 nt, whereas the SYSU100 construct generated six products ranging from 100 to 600 nt. The experimentally observed ladder patterns were reproducible using either an in-house or commercial T7 transcription system. A 500 μL in-house transcription preparation of either SYSU25 or SYSU100 provided sufficient material for approximately 50 analytical gel lanes. We further prepared synthetic ssDNA marker sets (S6D and M4D) and calibrated the S6D ladder against RNA standards of known length on 10%, 12%, 15%, and 20% Urea-PAGE gels. This calibration demonstrated that ssDNA and RNA do not migrate solely according to nucleotide length and provided gel-dependent RNA-equivalent size assignments for the S6D bands within the calibrated range. Together, the dual-ribozyme RNA ladders and RNA-calibrated ssDNA markers provide a low-cost reference system for the analysis of short- to medium-length RNA.

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Cite

BibTeXRIS
BibTeX
@article{zhao2026dualribozyme,
  title = {{The Dual-Ribozyme RNA Ladder and RNA-Calibrated ssDNA Marker System for Inexpensive RNA Size Standards}},
  author = {Yingying Zhao and Jiaqi Lin and Mengxiao Li and Lin Huang and Yuting Xie and Jia Wang},
  journal = {ACS Omega},
  year = {2026},
  month = oct,
  doi = {10.1021/acsomega.6c08063},
  url = {https://pubs.acs.org/acsodf/article/11/41/63792/5444625/The-Dual-Ribozyme-RNA-Ladder-and-RNA-Calibrated},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - The Dual-Ribozyme RNA Ladder and RNA-Calibrated ssDNA Marker System for Inexpensive RNA Size Standards
AU  - Yingying Zhao
AU  - Jiaqi Lin
AU  - Mengxiao Li
AU  - Lin Huang
AU  - Yuting Xie
AU  - Jia Wang
PY  - 2026
DA  - 2026/10/05
JO  - ACS Omega
DO  - 10.1021/acsomega.6c08063
UR  - https://pubs.acs.org/acsodf/article/11/41/63792/5444625/The-Dual-Ribozyme-RNA-Ladder-and-RNA-Calibrated
AB  - RNA size markers are essential for denaturing Urea-PAGE analysis, but commercial RNA ladders are expensive, available only in fixed size ranges, and require careful handling to prevent RNA degradation. Here, we developed a modular dual-ribozyme platform for preparing RNA size standards from single T7 transcription templates. Each construct contains a central RNA segment flanked by hammerhead and HDV ribozymes, enabling cotranscriptional self-cleavage to generate a predictable set of RNA products. The SYSU25 construct reproducibly produced short RNA marker bands at 25, 50, and 75 nt, whereas the SYSU100 construct generated six products ranging from 100 to 600 nt. The experimentally observed ladder patterns were reproducible using either an in-house or commercial T7 transcription system. A 500 μL in-house transcription preparation of either SYSU25 or SYSU100 provided sufficient material for approximately 50 analytical gel lanes. We further prepared synthetic ssDNA marker sets (S6D and M4D) and calibrated the S6D ladder against RNA standards of known length on 10%, 12%, 15%, and 20% Urea-PAGE gels. This calibration demonstrated that ssDNA and RNA do not migrate solely according to nucleotide length and provided gel-dependent RNA-equivalent size assignments for the S6D bands within the calibrated range. Together, the dual-ribozyme RNA ladders and RNA-calibrated ssDNA markers provide a low-cost reference system for the analysis of short- to medium-length RNA.
SN  - 2470-1343
ER  -