Laura M. Hertz; Julius B. Lucks

doi:10.21769/BioProtoc.5804

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Peer-reviewed

Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences

LH Laura M. Hertz ^§

JL Julius B. Lucks email

(§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: 102

评审: Alba BlesaAnonymous reviewer(s)

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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

Intrinsic termination

In vitro transcription

RNA-seq

NGS

Bioinformatics

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 GGTGCCCGAGTCAG	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·In^TM 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: S1000^TM)

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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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

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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.