Background

In the rapidly evolving fields of mRNA vaccine and RNA therapeutics development, ensuring the integrity of nucleic acids is critical. Nuclease contamination—whether from DNases or RNases—can degrade DNA or RNA, compromising the accuracy of research results and the yield of manufacturing processes. These enzymes are found everywhere: in laboratory environments, consumables, reagents, and even biological samples.

Due to the fragility of RNA molecules, detecting and eliminating nuclease activity is an essential part of mRNA synthesis quality control. However, traditional detection methods such as UV spectrophotometry, gel electrophoresis, or HPLC often lack the sensitivity, speed, or throughput needed for modern production demands.

To meet these challenges, The Hzymes DNase Assay Kit (Fluorescence) offers a fast, highly sensitive, and quantitative solution for detecting DNase contamination in laboratory environments and production processes.

Application

lt is used to detect DNase contamination in the environment and materials in scientific research and production processes.

Characteristics of Products

1. High Sensitivity: Limit of Detection is as low as 1.25 x10"U/uL (DNase l), which is only 1/8of the best competitor kit.

2. High precision: intra-batch CV<10%, inter-batch CV<15%Good stability: valid for one year at -20'c. Once the probe solution is prepared, split and stored at -20°c.

Precision and Stability of Hzymes DNase Assay Kit

Sensitivity higher than gel electrophoresis method

The Hzymes DNase Assay kit was in comparison with the nucleic acid hydrolysis-gel electrophoresis method, the same low-concentration enzyme samples were measured as shown in the figure:

Hyzmes DNase Assay kit measures 3x10 5 U/uL DNase l, which can be clearly distinguished from 0

Determination of DNase l by electrophoresis method, No significant difference between 9th/l0th channels (3x105U/uL) and 1st ~8th channels(≤3x10 U/uL), 17th channel (negative control).

Sensitivity: Hzymes DNase Assay kit VS competitor kit

Compared with one of the best competitor kits (fluorescence), the limit of detection of Hzymes DNase Assay kit is only 1/8 of that of the competitor kit.

When the sample concentration is 6.25 x10^-6 U/μL (low-value sample),the signal intensity of the competitor's DNase detection kit is indistinguishable from the negative control.

When the sample concentration is 6.25 x10^-6 U/uL (low-value sample), the signal intensity of the Hzymes DNase detection kit is clearly distinguishable from the negative control.

When the sample concentration of DNase l is from 1x10^-6 to 10x10^-6 U/μL (low-value sample), the Signal-to-voise(S/N)values of the competitor's DNase detection kit is close to l, however the signal intensity of Hzymes’ DNase Assay Kit is positive correlated to the concentration of DNase l.

Composition of the Hzymes DNase Assay Kit

Note: The standard is DNase l. The unit for DNase l activity is defined as the amount of enzyme that completely degrades lug of pBR322 DNA in DNase l reaction buffer at 37'c for 10 minutes ^[1]; one DNase l activity unit is equivalent at 0.3 Kunitz units ^[2].

Q&A

01: What is the reaction temperature and time of the DNase Assay kit?

Please incubate the reaction system at a constant temperature of 37 'C for 30 minutes.

Q2: When could false positive or false negative results or inaccurate quantitative results occur?

a.     Gel buffers, high concentration of viscous substances, surfactants and dark solutions may interfere with the luminescence of fluorophores;

b.     lf the sample solution to be tested contains substances that inhibit DNase activity, the result of the measurement is the overall enzyme activity of the sample solution, not the activity of the enzymes within it. These substances include:

l  High ionic strength solutions (eg.5M NaCl, 20x SSC, 3M sodium acetate, etc.)

l  Buffers with pH<4 or pH>9

l  Chaotropic agents, detergents, chelating agents or any solution that denatures proteins (eg. SDS, guanidine thiocyanate, urea, EDTA, etc.)

c.      Solutions that cause chemical instability of DNA probes, such as pH>9 solutions, caustic solutions (strong acids and bases, bleach. etc.)

Q3: While preparing standard solutions, in the pre-treatment steps why use the standard dilution buffer instead of DNase & RNase-free water to dilute?

Because the standard is relatively stable in the standard dilution buffer even after series dilution. But lf diluted with water, the activity of the standard may change during the series dilution process, resulting deviation of the standard.

Q4: How to adjust the appropriate microplate reader’s gain value?

Select auto-gain option if possible. lf your microplate reader does not have auto-gain selection option, first set the middle value according to the gain value range; then adjust the gain value according to the fluorescence signal of the positive control (79 uL DNase &RNase-free water + 1 u DNase l standard): reduce the gain value if the signal exceeds the upper limit of the instrument: Or increase the gain value appropriately if it is far below the upper limit of the instrument.

05: Why seriously contaminated samples may lead to false negative results?

The criterion for judging RNase contamination is: RFU30 (sample to be tested)> 2xRFU0(sample to be tested), that is, the RFU value at 30 minutes of reaction is more than twice the RFU value at 0 minute. lf the sample is seriously contaminated, it is possible that the reaction starts very quickly, so a very high RFU0 value is measured in a very short time, resulting in a false negative result of RFU30 (sample to be tested) ＜2 x RFU0 (sample to be tested) . At this time, sample to be tested needs to be diluted with DNase & RNase-free water.

06: lf the RFU value of the negative control is not 0, does it mean that the negative control is contaminated?

Uncertain. The negative control can also be detected with a low RFU signal (baseline value) but the value will not increase significantly along with the proceeding ofthe reaction.

Generally, it is considered that the RFU30 is less than twice the RFU0 which indicates that the negative control is not contaminated.

Ordering

References

[1] New England Biolabs DNase l User Guide

[2] Kunitz M. Crystalline Desoxyribo - nuclease l. lsolation and General Pro - perties Spectrophotometric Method for the Measurement of Desoxyribo nuclease Activity[J]. The Journal of General Physiology, 195033(4):349-362.