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Cat. No.: AlkB-200 (for 200U)

Cat. No.: AlkB-1k (for 1KU)

Cat. No.: AlkB-5k (for 5KU)

Description

AlkB (RNA/DNA demethylase, Nuclease-free) is a high-quality recombinant enzyme obtained through expression and purification in Escherichia coli, without DNase and RNase contamination. AlkB is an α-ketoglutarate (α-KG) and Fe(II)-dependent dioxygenase that efficiently repairs various alkylated damages in single-stranded or double-stranded DNA and RNA. It is commonly used to remove methylated modifications such as m6A in RNA, as well as m1A, m3C, m6A, and other modifications in DNA, for subsequent qPCR or high-throughput sequencing analysis.

N6-Methyladenosine (m6A) is the most prevalent and abundant RNA post-transcriptional modification in eukaryotes, present in various eukaryotic or viral RNAs, including mRNA, rRNA, tRNA, snRNA, microRNA (miRNA), and long non-coding RNA (lncRNA). This modification is believed to account for over 80% of RNA methylation modifications. The abundance of m6A in mRNA transcripts influences various RNA metabolic processes such as splicing, nuclear export, translation capacity, stability, and transcription.

AlkB can catalyze the demethylation of m6A in RNA in vitro using α-ketoglutarate and Fe(II) as cofactors, generating a 6-hydroxymethyl derivative (HO-m6A) that further converts to adenine.

AlkB is an adaptive stress response protein produced by bacteria exposed to methylating agents. It not only repairs methylated damages in RNA but also repairs methylated damages in DNA. The demethylation of 1-methyladenine (m1A) and 3-methylcytosine (m3C) in DNA by AlkB requires the involvement of O2, Fe2+, and α-ketoglutarate. During the demethylation of DNA, AlkB converts α-ketoglutarate into succinate and CO2, generating an unstable hydroxymethyl group that is released as formaldehyde. Single-stranded DNA (ssDNA) is more susceptible to methylation than double-stranded DNA (dsDNA), particularly at adenine N1 and cytosine N3. Therefore, AlkB is more efficient in repairing damages in ssDNA compared to dsDNA.

During RNA high-throughput sequencing library preparation, modified nucleotides in RNA can interfere with reverse transcription, leading to premature termination or transcription errors. Therefore, prior to reverse transcription, treating RNA with AlkB to demethylate potential 1-methyladenine (m1A), 3-methylcytosine (m3C), or 1-methylguanine (m1G) modifications is performed. This method, known as AlkB-facilitated RNA methylation sequencing (ARM-Seq), significantly enhances the sensitivity and specificity of RNA methylation sequencing, revealing more undiscovered small RNAs. PANDORA-seq is another high-throughput sequencing method that utilizes AlkB to discover additional unknown small RNAs.

Our provided AlkB is RNase-free, making it suitable for demethylation modifications, library preparation, and high-throughput sequencing. In publications, for example, in the PANDORA-seq method, it is noted that the expression-purified AlkB used in the study contained RNase, which could have a negative impact on high-throughput sequencing of RNA.

Application

Demethylation of m6A, m1A, m3C, or m1G in RNA; ARM-seq, PANDORA-seq, etc.; Demethylation of m1A, m3C, and m6A in DNA.

Enzyme Activity Definition

In a 20 µl reaction system of this product, the amount of AlkB enzyme that removes approximately 90-100% of m6A modification from 1 µg of HEK 293T total RNA is defined as 1 unit of activity. Under conditions where AlkB enzyme activity is well maintained, the activity of 1 µg of AlkB is approximately 1 unit.

Source

Recombinant protein expressed in Escherichia coli.

Purity

Free of DNases, RNases, and phosphatases.

Enzyme Storage Solution

200 mM NaCl, 2 mM DTT, 50% (v/v) Glycerol, 20 mM Tris-HCl (pH 8.0).

Inactivation or Inhibition

Adding an equal volume of buffer containing 11 mM EDTA and 200 mM ammonium acetate can terminate the reaction, or phenol-chloroform extraction can be performed.

A 200 U, 1000 U, or 5000 U packaging of this product can process approximately 200, 1000, or 5000 RNA samples of around 1 µg each. If a more thorough reaction is desired, the amount of enzyme added needs to be increased accordingly, and the number of samples that can be processed will be reduced accordingly.

Precautions

• During use, it is recommended to keep the product in an ice box or on an ice bath. After use, it should be immediately stored at -20°C.
• This product is intended for scientific research purposes by professionals only. It is not intended for clinical diagnosis or treatment, and should not be used for food or drug purposes. It should not be stored in a regular household setting.
• For your safety and health, please wear lab attire and disposable gloves when handling the product.

Storage

The minimum shelf life is 1 year at -20°C.

Related:

• AlkB (RNA/DNA Demethylase, Nuclease-free)
• AlkB (D135S) (RNA m1G Demethylase, Nuclease-free)
• AlkB (D135S/L118V) (RNA m22G Demethylase, Nuclease-free)