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Cat. No.: AlkBDL-50 (for 50U)

Cat. No.: AlkBDL-250 (for 250U)

Cat. No.: AlkBDL-1250 (for 1250U)

Description

AlkB (D135S/L118V) (RNA m22G Demethylase, Nuclease-free) is a high-quality recombinant enzyme, expressed and purified from E. coli, free from DNase and RNase.

AlkB is an α-Ketoglutarate (α-KG) and Fe(II)-dependent dioxygenase that effectively repairs various alkylation damages in single-stranded or double-stranded DNA and RNA. It is commonly used to remove methylation modifications such as m6A in RNA, and m1A, m3C, and m6A in DNA, facilitating subsequent qPCR or high-throughput sequencing analysis.

AlkB (D135S/L118V) (RNA m22G Demethylase, Nuclease-free) is a mutant based on AlkB (RNA/DNA Demethylase, Nuclease-free) (R0639), where the 135th aspartic acid (Asp, D) is mutated to serine (Ser, S) and the 118th leucine (Leu, L) is mutated to valine (Val, V). AlkB (D135S/L118V) is more efficient than wild-type AlkB in removing N2,N2-dimethylguanosine (m22G) methylation modifications in transfer RNA (tRNA), aiding subsequent qPCR or high-throughput sequencing analysis. While m2G has minimal impact on Watson-Crick base pairing, m22G, like m1A, m1G, and m3C, is a modification that hinders base pairing, causing termination or mutations during reverse transcription. Removing one methyl group from m22G to form N2-methylguanosine (m2G) enables efficient transcription into cDNA using appropriate thermostable reverse transcriptase for subsequent qPCR or high-throughput sequencing.

N2,N2-dimethylguanosine (m22G) is a characteristic nucleotide with two methyl groups on its external amine, found in over 80% of eukaryotic species' tRNAs at the 26th position (G26) in the bend between the D-stem and the anticodon stem. m2G and m22G are common modifications in cellular RNA.

tRNA is the most extensively modified RNA family in cells, playing a critical role in cellular physiology. Modifications in the anticodon loop are crucial for fine-tuning mRNA translation, while those outside the anticodon loop contribute to tRNA stability, folding, localization, and quality control. However, methylation modifications like m1A, m1G, m3C, and m22G can hinder or block cDNA synthesis during reverse transcription of tRNA or tsRNA, and stable secondary structures can interfere with adapter ligation. Efficient removal of these methylations is essential for reducing their negative effects on reverse transcription and ligation, ensuring better adapter ligation and reverse transcription outcomes.

During RNA high-throughput sequencing library preparation, modified nucleotides in RNA can interfere with reverse transcription, leading to transcription termination or errors. Using AlkB to demethylate RNA containing m1A, m1G, or m3C before high-throughput sequencing, a method known as AlkB-facilitated RNA methylation sequencing (ARM-Seq), significantly enhances the sensitivity and specificity of sequencing methylated RNA, revealing previously undiscovered small RNAs. Techniques like PANDORA-seq, DAMM-seq, DM-TGIRT-seq, and CPA-seq utilize AlkB or its mutants for demethylation, enabling accurate and efficient high-throughput sequencing of methylated RNAs like tRNA or tsRNA.

To achieve optimal sequencing of methylated RNAs, it is recommended to use AlkB (RNA/DNA Demethylase, Nuclease-free) (R0639) in conjunction with AlkB (D135S) (RNA m1G Demethylase, Nuclease-free) (R0641) and AlkB (D135S/L118V) (RNA m22G Demethylase, Nuclease-free) (R0643) to simultaneously remove multiple methylations, including m1A, m3C, m6A, m1G, and m22G.

Our produced AlkB is free from RNase, making it ideal for demethylation and subsequent library preparation and high-throughput sequencing.

RNase/DNase Activity Detection

The product was incubated with RNA or DNA at 37ºC for 100 minutes, and no significant RNase/DNase activity was detected.

Applications

• Demethylation of m22G in tRNA

• DAMM-Seq

• ARM Seq

• PANDORA-seq

• DM-TGIRT-seq

• CPA-seq

Enzyme Activity Definition

In a 100µl reaction system, the amount of AlkB (D135S/L118V) enzyme required to remove the m22G modification from 1nmol of tRNA to generate m2G is defined as one activity unit. Approximately 4µg of AlkB (D135S/L118V) has an enzyme activity of about 1U when maintaining good enzyme activity.

Source

Recombinant protein expressed in E. coli.

Purity

Free from DNases, RNases, and phosphatases.

Enzyme Storage Solution

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

Inactivation or Inhibition

The reaction can be terminated by adding 5mM EDTA or by phenol-chloroform extraction.

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)