Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences
进化筛选获得的转录型核糖开关序列的大规模并行体外功能分析
(§Technical contact: laurahertz2027@u.northwestern.edu) 发布: 2026年09月05日第16卷第17期 DOI: 10.21769/BioProtoc.5804 浏览次数: 119
评审: Alba BlesaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2026
Abstract
Riboswitches are structured non-coding RNA elements that regulate gene expression in response to small molecules; they serve as valuable systems in both public health and biophysical research by elucidating principles around RNA–ligand interactions, structure, and cellular function. Traditional approaches to studying riboswitches have relied on low-throughput techniques such as reporter assays or gel electrophoresis analysis of transcriptional products, which are limited in scalability. In this study, we present a high-throughput protocol to characterize the transcriptional activity of nearly 2,000 natural variants of the fluoride riboswitch in in vitro transcription. Starting with bioinformatics, we compiled a comprehensive dataset of riboswitch variants and then employed massive parallel oligonucleotide synthesis to generate an oligo pool of the riboswitch library. This pool was transcribed in vitro, converted into an Illumina-compatible next-generation sequencing (NGS) library, and analyzed to identify transcriptionally active riboswitch candidates. The workflow integrates natural riboswitch bioinformatic acquisition into a quantitative readout in a single streamlined pipeline, enabling large-scale exploration of transcriptional riboswitch function. This protocol offers a scalable method for mapping genotype-to-function relationships across transcriptional riboswitch families, accelerating the identification of functional variants for desired applications.
Key features
• Bioinformatics to acquire full sequences (aptamer + downstream expression platform) of naturally occurring riboswitches.
• E. coli RNA polymerase in vitro transcription to characterize nearly 2,000 fluoride riboswitches and analysis with NGS.
Keywords: Riboswitches (核糖开关)
Intrinsic termination (内在转录终止)
In vitro transcription (体外转录)
RNA-seq (RNA 测序)
NGS (二代测序)
Bioinformatics (生物信息学)
Graphical overview
Assessing the riboswitch functionality in an in vitro–transcribed context of known aptamers. This figure is adapted from Hertz et al. (2026) NAR [1].
Background
Riboswitches are important non-coding regulatory elements found across all kingdoms of life that have broad applications in public health [2] and as biophysical models [3]. Riboswitches contain two structural and functional domains: the aptamer domain that binds a ligand, and the expression platform that carries out the gene regulation mechanism. Structurally, the aptamer domains of riboswitches are highly evolutionarily conserved in order to carry out the singular role of ligand binding. Expression platforms are generally poorly conserved, as there are examples that can carry out different forms of gene regulation, including at the transcriptional [4], translational [5], degradation [6], and splicing levels [7]. As a result, there is relatively poor characterization of the function of all known riboswitches within a particular aptamer class. To fully explore riboswitches as genetic control elements in mechanistic and molecular tool development, we need high-throughput methods to characterize expression platform function.
Next-generation sequencing (NGS) is a powerful high-throughput method that can be applied to quantify riboswitch regulatory function. At the cellular level, RNA-seq data validates active riboswitches [8]. In synthetic systems, NGS can reveal optimal sequences to generate novel transcriptional riboswitches (histamine, tetR, etc.) or reveal translational sequence dependence [9,10]. Similarly, researchers characterized the self-cleavage activity of 2,625 natural Twister ribozymes [11]. We sought to characterize the computationally predicted fluoride aptamer sequences deposited on Rfam [12] as full fluoride riboswitches in a synthetic transcription environment to identify transcriptionally active variants. We employed bioinformatics to compile the riboswitch sequences and massively parallel oligo synthesis to generate the pool of riboswitch variants. Then, we performed transcription, generated an Illumina NGS library, and analyzed the output reads to show that this high-throughput pipeline is fit for identifying high-performing transcriptional riboswitch candidates.
Materials and reagents
Biological materials
1. Oligo pool (Twist Biosciences)
2. Oligo sequences (Integrated DNA technologies, IDT)
| Name | Sequence | Supplier | Order specifications |
|---|---|---|---|
| Primer A | gcttccggcttgattctaaagatc | IDT | PAGE purified |
| Primer B | cggacagaaaatttgtgccc | IDT | PAGE purified |
| Linker | /5Phos/rCrUrGrArCrUrCrGrGrGrCrArCrCrArArGrGrA/3ddC/ | IDT | Standard desalting |
| Primer C | /5BiosG/GTCCTTGGTGCCCGAGT | IDT | Standard desalting |
| SS2.0 Dumbbell | /5Phos/TGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG/3SpC3/ | IDT | PAGE purified |
Illumina INDEX Primers (Illumina Knowledge Article #7129) | CAAGCAGAAGACGGCATACGAGAT[INDEX]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGAACAGCGAC TAGGCTCTTCA | IDT | PAGE purified |
| Primer D | CTTTCCCTACACGACGCTCTTCCGATCTYYYRGTCCTT GGTGCCCGAG*T*C*A*G | IDT | Standard desalting |
| TruSeq universal adapter | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT | IDT | Standard desalting |
Reagents
1. RNase/DNase-free water (Invitrogen, catalog number: 10977-015)
2. dNTPs (NEB, catalog number: N0447L)
3. 5× Q5 reaction buffer (NEB, catalog number: B9027S)
4. Q5 polymerase (NEB, catalog number: M0491L)
5. Bead Purification kit (Cytiva, catalog number: 29343052)
6. Tris (Sigma, catalog number: T3253-500G)
7. EDTA pH 8.0 (Invitrogen, catalog number: 15575020)
8. KCl (Sigma-Aldrich, catalog number: P9541-500G)
9. DTT (Invitrogen, catalog number: 18090010); create 10-μL single-use aliquots and store at -20 °C
10. MgCl2 (NEB, catalog number: B0510A)
11. BSA (NEB, catalog number: B9000S)
12. E. coli RNAP holoenzyme (NEB, catalog number: M0551S)
13. NTPs (Fisher Scientific, catalog number: FERR1481)
14. Rifampicin (Sigma-Aldrich, catalog number: R3501-250MG)
15. NaCl (Sigma-Aldrich, catalog number: S3014-1KG)
16. TRIzol (Ambion, catalog number: 15596018)
17. Chloroform (Acros Organics, catalog number: 423555000)
18. GlycoBlue (Invitrogen, catalog number: AM9515)
19. Isopropanol (Sigma-Aldrich, catalog number: 190764-1L)
20. Ethanol (Sigma-Aldrich, catalog number: 459844-500ML)
21. 10× TURBO DNase buffer (Invitrogen, catalog number: 4022G)
22. TURBO DNase (Invitrogen, catalog number: AM2238)
23. 50% PEG800 (NEB, catalog number: 1004S)
24. 5′ Adenylation kit (NEB, catalog number: E2610S)
25. 10× T4 RNA ligase buffer and T4 RNA ligase, Trunc KQ (NEB, catalog number: M0373L)
26. SUPERase·InTM RNase inhibitor (Invitrogen, catalog number: AM2696)
27. NaOAc (Invitrogen, catalog number: AM9740)
28. SSIV buffer and SSIV enzyme (Invitrogen, catalog number: 18090010)
29. NaOH (Sigma, catalog number: S5881)
30. HCl (Sigma, catalog number: 320331-500ML)
31. Betaine (Sigma, catalog number: B0300-1Vl)
32. T4 DNA ligase buffer and ligase enzyme (NEB, catalog number: B0202S)
33. ExoI (NEB, catalog number: M0293S)
34. High-sensitivity Qubit assay (Invitrogen, catalog number: Q32854)
35. DNA spin columns (EconoSpin, catalog number: 1910-250)
36. Buffer PB (Qiagen, catalog number: 19066)
37. Buffer PE (Qiagen, catalog number: 19065)
Solutions
1. 10× transcription buffer (see Recipes)
2. Transcription start/stop solution (see Recipes)
Recipes
1. 10× transcription buffer
| Reagent | Final concentration | Volume |
|---|---|---|
| RNase/DNase-free water | n/a | 448 μL |
| Tris pH 8.0 | 200 mM | 200 μL |
| EDTA pH 8.0 | 1 mM | 2 μL |
| KCl | 500 mM | 250 μL |
| DTT (fresh or a one-use frozen aliquot) | 10 mM | 100 μL |
| Total | n/a | 1 mL |
Note: For convenience, create a stock solution of the 10× TB without DTT and store at room temperature. During setup for the transcription reaction, prepare a fresh DTT solution or thaw a one-use aliquot (stored at -20 °C).
2. Transcription start/stop solution
| Reagent | Final concentration | Volume |
|---|---|---|
| RNase/DNase-free water | n/a | 15 μL |
| NTPs | 5 mM | 4 μL |
| Rifampicin | 0.1 mg/mL | 1 μL |
| Total | n/a | 20 μL |
Note: Rifampicin is for performing a single round of transcription [13]. If multiple rounds of transcription initiation to have multiple RNA copies from one DNA template are desired, then replace rifampicin with water.
Laboratory supplies
1. 1.7 mL tubes (Corning, catalog number: MCT-175-C)
2. 0.7 mL tubes (Bio Plas, catalog number: 4040)
3. PCR tubes (Eppendorf, catalog number: 951-01-002-2)
4. Magnetic stand for bead purification (Ergi Lab Supplies, catalog number: 1008)
5. QIAquick PCR Purification kit (50) (Qiagen, catalog number: 28104)
6. 20 μL pipette tips (Rainin, catalog number: 30389226)
7. 200 μL pipette tips (Rainin, catalog number: 30389239)
8. 1,000 μL pipette tips (Rainin, catalog number: 30389212)
Equipment
1. Thermocycler (Bio-Rad, model: S1000TM)
2. Benchtop centrifuge (Eppendorf, model: EP5405000441)
3. Bioanalyzer (Agilent, model: 2100 bioanalyzer)
4. Freezer (-20 °C) (Thermo Scientific, model: MF02PA-SAEE-TS)
5. Qubit (Thermo Scientific, catalog number: Q33239)
6. Pipettes 2, 20, 200, 1,000 μL (Rainin, catalog number: 30579367)
7. Sequencing gel apparatus (Bio-Rad, catalog number: 165-3860)
Software and datasets
| Type | Software/dataset/resource | Version | Date | License | Access (free or paid) |
|---|---|---|---|---|---|
| Code | GitHub, Code S1 | 0.0 | Jan 2026 | GNU v3.0 | Free [14] |
| Data | Rfam | 15.0 | Sep 2024 | CC0 | Free [12] |
| Software | BacTermFinder | 1.0 | Dec 2024 | GPL-3.0 | Free [15] |
| Software | PEAR | 0.9.6 | Mar 2014 | CCPL | Free [16] |
| Software | megahit | 1.0.6.1 | Jun 2017 | GPL-3.0 | Free [17] |
| Software | STAR | 2.7.9a | May 2021 | MIT | Free [18] |
Procedure
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文章信息
稿件历史记录
提交日期: May 25, 2026
接收日期: Jul 22, 2026
在线发布日期: Aug 13, 2026
出版日期: Sep 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Hertz, L. M. and Lucks, J. B. (2026). Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences. Bio-protocol 16(17): e5804. DOI: 10.21769/BioProtoc.5804.
Download Citation in RIS Format
分类
生物信息学与计算生物学
分子生物学 > RNA > 转录
微生物学 > 微生物遗传学 > RNA
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