Oct 30, 2025
Heliscreener RNA Unwinding Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Heliscreener RNA Unwinding Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrwr53lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 30, 2025
Last Modified: October 30, 2025
Protocol Integer ID: 228582
Keywords: assay technical manual the heliscreener rna, heliscreener rna, activity of rna helicase, rna helicase, unwinding assay, stranded rna, unwinding assay technical manual, rna, biochemical assay, inhibitor ic50 determination, accurate enzyme kinetic, dsrna, assay, enzyme reaction
Abstract
The Heliscreener RNA Unwinding Assay is a biochemical assay designed to measure the RNA unwinding activity of RNA helicases in the DExH/D-box family. The assay utilizes a labeled double-stranded RNA (dsRNA) substrate that is quenched when intact and emits far-red fluorescence following unwinding. The assay can be performed in kinetic (continuous monitoring) or endpoint mode. Kinetic mode is recommended for initial velocity measurements, allowing for accurate enzyme kinetics and inhibitor IC50 determinations. Alternatively, for screening, the enzyme reaction can be stopped by adding the Stop 26 Detect Buffer B, and the quenched reactions can be read in endpoint mode.
Materials
Product Specifications:
- Heliscreener RNA Unwinding Assay
- 1,000 assays* (Part# 3051-1K)
- 10,000 assays* (Part# 3051-10K)
*NOTE: The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 384-well plates, using a 20 µL complete assay volume.
Materials Provided:
- Reporter RNA: 4 µM in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA. dsRNA with a 3’ overhang. The fluorescent strand is labeled with Cy5 and the quenching strand is labeled with Iowa Black Quencher.
- Capture RNA: 4 µM in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA. ssRNA complementary to the fluorescent strand in the Reporter RNA.
- ATP, 100 mM: 100 mM in Nuclease-free water.
- Enzyme Assay Buffer D, 10X: 500 mM Tris (pH 7.5), 20 mM MgCl2, and 0.1% Triton X-100. Optimized to support activity of DDX3, DDX5, DDX17, and DHX9.
- Unwound RNA Control: 4 µM RNAs in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA. A mixture of RNAs that mimics a completed unwinding reaction.
- Stop 26 Detect Buffer B, 10X: 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Quenches the Enzyme Reaction by chelating metals required for activity.
Materials Required or Recommended but Not Provided:
- Enzyme: Validated for use with purified DDX3, DDX5, DDX17, and DHX9, available from BellBrook as stand-alone products (Part# 2251, 2307, 2309, 2317).
- Ultrapure Nuclease Free Water: Use nuclease-free water such as: Invitrogen Part # AM9930.
- RNase Inhibitor (recommended): Such as RNaseOUT™ or RNasin®.
- Assay Plate: Use assay plates that are entirely black with a nonbinding surface. Recommended Corning® 384-well plates (Cat.# 4514).
Troubleshooting
Before start
- Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
- Ensure that your plate reader is capable of a) measuring Cy5 fluorescence (excitation peak: 649 nm, emission peak: 670 nm), b) maintaining a temperature of 30°C, and c) performing kinetic readings at an interval of one to three minutes. Running the assay at room temperature is possible but may require more enzyme, while a larger reading interval may result in missing the initial velocity phase of the enzyme reaction.
- Define the Maximum Signal Window for Your Plate Reader: The RFUs for the Unwound RNA Control (20 µL of 8 nM Unwound RNA Control in 0.5X Enzyme Assay Buffer D) represents the High signal, and the RFUs for the Reporter RNA (20 µL of 8 nM Reporter RNA in 0.5X Enzyme Assay Buffer D) represents the Low signal; Maximum Signal = RFUHigh – RFULow. Prepare High and Low RFU Controls in quantities sufficient to perform at least 12 replicates for each condition. Calculate the Z’ factor; values greater than 0.7 are acceptable.
Introduction
The Heliscreener RNA Unwinding Assay is a biochemical assay designed to measure the RNA unwinding activity of RNA helicases in the DExH/D-box family. The assay utilizes a labeled double-stranded RNA (dsRNA) substrate that is quenched when intact and emits far-red fluorescence following unwinding (see Figure 1).
The assay can be performed in kinetic (continuous monitoring) or endpoint mode. Kinetic mode is recommended for initial velocity measurements, allowing for accurate enzyme kinetics and inhibitor IC50 determinations. Alternatively, for screening, the enzyme reaction can be stopped by adding the Stop & Detect Buffer B, and the quenched reactions can be read in endpoint mode.
Key Specifications:
- Single-addition, mix-and-read format
- Excellent data quality (Z’ ≥ 0.7)
- Far red tracer minimizes interference from fluorescent compounds and light scattering
Key Applications:
- Screening for enzyme inhibitors/activators
- Generating dose-response curves and IC50 values for inhibitors
- Kinetic and mechanistic analyses
Figure 1. Schematic Overview of the Heliscreener RNA Unwinding Assay. Fluorescence is quenched when the Reporter RNA is intact. RNA helicases unwind the Reporter RNA, releasing the far-red fluorescence (Cy5) from the quencher (Iowa Black® Quencher). A Capture RNA (green) hybridizes to the fluorescent strand, preventing it from re-annealing with the quenching strand.
Product Specifications
| Product | Quantity | Part# | |
| Heliscreener RNA Unwinding Assay | 1,000 assays* | 3051-1K | |
| 10,000 assays* | 3051-10K |
*NOTE: The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 384-well plates, using a 20 µL complete assay volume.
Storage
Reporter RNAs and Capture RNA should be aliquoted for multiple uses and stored at -80°C.
Use the reagents provided in this kit within 6 months from the date of receipt.
Materials Provided
| Component | Composition | Notes | |
| Reporter RNA | 4 µM in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA | dsRNA with a 3’ overhang. The fluorescent strand is labeled with Cy5 and the quenching strand is labeled with Iowa Black Quencher. | |
| Capture RNA | 4 µM in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA | ssRNA complementary to the fluorescent strand in the Reporter RNA. | |
| ATP, 100 mM | 100 mM in Nuclease-free water | ||
| Enzyme Assay Buffer D, 10X | 500 mM Tris (pH 7.5), 20 mM MgCl2, and 0.1% Triton X-100 | Enzyme Assay Buffer D has been optimized to support activity of DDX3, DDX5, DDX17, and DHX9. | |
| Unwound RNA Control | 4 µM RNAs in 50 mM Tris, 50 mM NaCl, and 1 mM EDTA | A mixture of RNAs that mimics a completed unwinding reaction: the fluorescent strand annealed to the Capture RNA, and the quenching strand by itself. It serves as a control for the maximum assay window. | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer B components quench the Enzyme Reaction by chelating metals required for activity. |
Materials Required or Recommended but Not Provided
| Component | Notes | |
| Enzyme | The Heliscreener RNA Unwinding Assay is validated for use with purified DDX3, DDX5, DDX17, and DHX9, which are available from BellBrook as stand-alone products (Part# 2251, 2307, 2309, 2317). | |
| Ultrapure Nuclease Free Water | Some deionized water systems are contaminated with enzymes that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease-free water such as: Invitrogen Part # AM9930 | |
| RNase Inhibitor (recommended) | RNase inhibitors, such as RNaseOUT™ or RNasin®, provide protection against RNase contamination, ensuring that the detected fluorescent signal originates from the helicase enzyme reaction rather than from RNA degradation. A concentration greater than 0.005 U/µL of RNaseOUT or RNasin or equivalent is recommended in the enzyme reaction. If the RNase inhibitor is omitted, it is essential to include a no-ATP control to assess any RNase-related fluorescence. | |
| Assay Plate | It is important to use assay plates that are entirely black with a nonbinding surface. We recommend Corning® 384-well plates (Cat.#4514). The suggested plate has a square well top that enables easier robotic pipetting and a round bottom that allows good Z’ factors. It has a recommended working volume of 15–20 µL. |
Before You Begin
- Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
- Ensure that your plate reader is capable of a) measuring Cy5 fluorescence (excitation peak: 649 nm, emission peak: 670 nm), b) maintaining a temperature of 30°C, and c) performing kinetic readings at an interval of one to three minutes. Running the assay at room temperature is possible but may require more enzyme, while a larger reading interval may result in missing the initial velocity phase of the enzyme reaction.
- Define the Maximum Signal Window for Your Plate Reader: The RFUs for the Unwound RNA Control (20 µL of 8 nM Unwound RNA Control in 0.5X Enzyme Assay Buffer D) represents the High signal, and the RFUs for the Reporter RNA (20 µL of 8 nM Reporter RNA in 0.5X Enzyme Assay Buffer D) represents the Low signal; Maximum Signal = RFUHigh – RFULow. Prepare High and Low RFU Controls in quantities sufficient to perform at least 12 replicates for each condition. Calculate the Z’ factor using the equation below; values greater than 0.7 are acceptable.
Protocol
Performing a Kinetic Enzyme Assay
Figure 2. Simple Mix-and-Read Format. The plate is read every one to three minutes for a total of 30 minutes to capture the initial velocity and maximize the assay window.
The following assay protocol is for 384-well format, using 20 µL Complete Assay volume when the plates are read. All the reagent mixes can be prepared ahead of time and stored on ice for at least 2 hours before use.
1. Prepare Working Stocks (see Table 1)
a. Prepare Complete Assay Buffer containing 0.5X Enzyme Assay Buffer D and 0.005 U/µL of RNaseOUT (or equivalent of other RNase inhibitors, recommended) in Ultrapure Nuclease-Free Water.
b. Dilute your enzyme to 4X the desired concentration in Complete Assay Buffer.
c. Dilute 100 mM ATP to 4X the desired concentration in Complete Assay Buffer.
d. Prepare 2X RNA Mix by combining 16 nM Reporter RNA, and 16 nM Capture RNA in Complete Assay Buffer.
| Component | Stock | Working Concentration | Final Concentration in 20 µL Enzyme Reaction | |
| Enzyme* | 4X | 1X | ||
| ATP* | 100 mM | 4X | 1X | |
| Reporter RNA | 4 µM | 16 nM | 8 nM | |
| Capture RNA | 4 µM | 16 nM | 8 nM | |
| Enzyme Assay Buffer D | 10X | 0.5X | 0.5X | |
| RNaseOUT (recommended) | 0.005 U/µL | 0.005 U/µL |
Table 1. Kinetic Assay Components. *Typical Enzyme and ATP concentrations shown in Table 2.
2. Run Enzyme Reaction
a. Add 5 μL of 4X Enzyme to each well.
b. Add 5 μL of 4X ATP to each well.
c. Get the plate reader ready. Then, add 10 μL of 2X RNA Mix. Mix briefly on a plate shaker. Read every one to three minutes for a total of 30 minutes at 30°C.
Concentration in 20 µL Complete Assay
| Component | DDX3 | DDX5 | DDX17 | DHX9 | |
| Reporter RNA | 8 nM | 8 nM | 8 nM | 8 nM | |
| Capture RNA | 8 nM | 8 nM | 8 nM | 8 nM | |
| ATP* | 0.5 mM | 1.5 mM | 0.5 mM | 0.14 mM | |
| Enzyme Assay Buffer D | 0.5X | 0.5X | 0.5X | 0.5X | |
| RNaseOUT™ (recommended) | 0.005 U/µL | 0.005 U/µL | 0.005 U/µL | 0.005 U/µL |
*ATP is near the Km concentration for DDX3, DDX5, DDX17, and 2*Km concentration for DHX9
Table 2. Example Kinetic Assay Conditions for DDX3, DDX5, DDX17, and DHX9
Figure 3. Example Enzyme Titration – Kinetic Assay. For screening and profiling inhibitors, choose an enzyme concentration that maximizes signal window while still in linear range at the 5 min time point. For example, the ideal enzyme concentration in this data set is 80 nM, 5 nM, 2 nM, and 40 nM for (A) DDX3, (B) DDX5, (C) DDX17, and (D) DHX9 respectively.
Performing an End-Point Enzyme Assay
The assay can also be conducted as an end-point assay. However, it is important to note that the initial velocity develops quickly, even at low enzyme concentrations; therefore, the reaction time should be determined carefully. We have found that a reaction time of 15 minutes is ideal for validated enzymes, providing a flexible workflow while remaining close to the initial velocity phase of the reaction. The following protocol is designed for a 384-well format, utilizing 15 µL of enzyme reaction solution and 5 µL of Stop & Detect Buffer B. All reagent mixtures can be prepared in advance and stored on ice for up to 2 hours before use.
1. Prepare Working Stocks (see Table 3)
a. Prepare Complete Assay Buffer containing 0.5X Enzyme Assay Buffer D and 0.005 U/µL of RNaseOUT™ (or equivalent of other RNase inhibitors, recommended) in Ultrapure Nuclease-Free Water.
b. Dilute your enzyme to 3X the desired concentration in Complete Assay Buffer.
c. Dilute 100 mM ATP to 3X the desired concentration in Complete Assay Buffer.
d. Prepare 3X RNA Mix by combining 24 nM Reporter RNA, and 24 nM Capture RNA in Complete Assay Buffer.
e. Dilute 10X Stop & Detect Buffer B to 4X in Ultrapure Nuclease-Free Water.
| Component | Stock | Working Concentration | Final Concentration in 15 µL Enzyme Reaction | |
| Enzyme | 3X | 1X | ||
| ATP | 100 mM | 3X | 1X | |
| Reporter RNA | 4 µM | 24 nM | 8 nM | |
| Capture RNA | 4 µM | 24 nM | 8 nM | |
| Enzyme Assay Buffer D | 10X | 0.5X | 0.5X | |
| RNaseOUT™ (recommended) | 0.005 U/µL | 0.005 U/µL | ||
| Stop & Detect Buffer B | 10X | 4X | 1X in 20 µL Complete Assay |
Table 3. End-point Assay Components.
2. Run Enzyme Reaction
a. Add 5 μL of 3X Enzyme to each well.
b. Add 5 μL of 3X ATP to each well.
c. Add 5 μL of 3X RNA Mix. Mix briefly on a plate shaker. Incubate at 30°C for 15 minutes.
d. Add 5 μL of 4X Stop & Detect Buffer B. Mix on a plate shaker. Read the plate.
Performing Single Compound Screening and Dose-Response Assays
Single Compound Screening and Dose-Response Assays follow the protocol listed in Section 4.1 or 4.2. The target enzyme is added to the test compounds pre-dispensed in wells; the total mixture volume should be 5 µL. The final concentration of the test compounds should be based on the volume of the enzyme reaction (20 µL for kinetic assay and 15 µL for end-point assay). We recommend mixing gently on a plate shaker for 40 to 60 seconds and preincubating for 30 minutes at room temperature to allow equilibration of the E-I complex.
Figure 4. Example Dose Response Curve. (A) A kinetic assay using 2 nM DDX17 and varying amounts of the probe compound, Suramin. (B) The initial velocity phase of the progression curves was fitted linearly to obtain slopes that represent the enzyme's initial rate at each compound concentration. (C) These slopes are then plotted against compound concentration for accurate IC50 determination. (D) A dose-response curve was obtained with an endpoint assay using 2 nM DDX17 for 15 minutes; the higher value relative to the kinetic assay (C) is a result of exceeding initial velocity conditions in the endpoint assay.
Using the Assay with Different Volumes and Plate Formats
Please check the working plate volumes from the manufacturer to ensure they are within the suggested volume ranges of your plate.
| Component | Total Volume | Enzyme Reaction Volume | 2X RNA Mix Volume | |
| 96 Well Low Volume Plate | 50 µL | 25 µL | 25 µL | |
| 384 Well Low Volume Plate | 20 µL | 10 µL | 10 µL | |
| 1536 Well Low Volume Plate | 8 µL | 4 µL | 4 µL |
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