Direct Substrate of T4 NDA/RNA Ligase

In the presence of ATP, T4 DNA/RNA ligase can use ATP to add 5',5'-adenyl pyro-phosphoryl moiety (5'-app) to the 5' end of a nucleic acid chain. Then the 3'-OH of the other nucleic acid chain attacks this app linkage, and finally the two nucleic acid chains are connected. The synthetic 5'-adenylated oligonucleotide can accomplish this process and does not require the involvement of ATP.

Fig.1 The connecting process of two nucleic acid chains by T4 DNA/RNA ligase. (CD BioGlyco)

Typical Applications of 5'-Adenylated Oligonucleotide

5'-Adenylated oligonucleotides for linking single-stranded DNA/RNA are widely used in next-generation sequencing (NGS). In microRNA (miRNA) analysis, ATP is not required for the connection of the target molecule (miRNA) to the adenylated oligonucleotide, thus minimizing potential self-ligation and greatly simplifying the nucleic acid library preparation process. Conventionally, adenylation-based oligonucleotide modification can be achieved by chemical synthesis or enzymatic modification.

Adenylation-based Oligonucleotide Modification Service at CD BioGlyco

CD BioGlyco offers custom adenylation-based oligonucleotide synthesis services to clients. We synthesize 5'-adenylated oligonucleotide sequences and provide other Oligonucleotide Modification Services (in addition to heat-sensitive modifications) to support customers in accessing unique modified oligonucleotides for direct use with RNA/DNA ligases. Our approach allows for a cost-effective scale of your customized oligonucleotides and secures your application in developing commercial analyses with fixed linker sequences.

Fig.2 Chemical structure of 5'-adenylation app. (CD BioGlyco)

To avoid self-ligation of 5 'adenylation oligonucleotides, the synthesized sequence carries a blocking group at the 3' end such as a C3 propyl spacer. As a result, our nucleotides are stable enough to be applied in any link-based analysis.

• Constructing miRNA library: It is extremely difficult to construct a high-quality miRNA library from cellular RNA. T4 RNA ligase will lead to a large amount of self-ligation in the presence of ATP. This difficulty can be avoided by attaching a 5'-app-modified adaptor to the 3'-OH of the miRNA (no ATP involved) and then linking the 5'-end of the miRNA to another adaptor (ATP involved). Through this process, both ends of the miRNA are attached with adaptors, which can be further cloned to the surface of the carrier for sequencing.
• Working as activated substrates of nucleic acid for in vitro-selected ribozymes/deoxyribozymes: In vitro selection techniques have expanded our knowledge of the nucleic acid function and provided new reagents with potential therapeutic and diagnostic applications. In vitro-selected ribozymes/deoxyribozymes have high connection efficiency and a wide connection range. 5'-App-modified oligonucleotides are specific active substrates for these enzymes, and constructing required long, modified RNA molecules by them is a useful strategy.
• Increasing possible RNA substrates for ribozymes/deoxyribozymes: 5'-App-modified RNA has a similar structure to 5'-triphosphorylated RNA (5'-ppp RNA), which is the product of T7 RNA polymerase in vitro transcription, and 5'-ppp NTP can only be 5'-ppp G. Therefore, if replacing 5'-ppp RNA with 5'-app-modified RNA, the possible RNA substrates for ribozymes/deoxyribozymes would be increased.
• Activating 5'-pyrimidine-rich RNA for ligation: Obtaining 5'-phosphorylated pyrimidine-rich RNAs is difficult. But 5'-adenylation of these RNA molecules is a good strategy to activate these molecules for ligation.
• Inserting 5'-end labeling: The adenosine group of the 5'-adenylated oligonucleotide has a diol structure in ribose, which can be oxidized to an aldehyde group. The obtained aldehyde group can be attached to functional groups such as fluorescent groups, which are labeled at the 5' end of the oligonucleotide.

Applications

• Construct miRNA library.
• Next generation sequencing.
• As activated substrates of nucleic acid for in vitro-selected ribozymes/deoxyribozymes.
• Broaden possible RNA substrates for ribozymes/deoxyribozymes.
• Activate 5'-pyrimidine-rich RNA for ligation.
• Insert 5'-end labeling on oligonucleotides.

Advantages

• 5'-Adenylated oligonucleotides with a 3'-blocker.
• Efficient synthesis and modification process.
• Large-scale production capacity.
• Support commercial assays.

CD BioGlyco is a scientist collective dedicated to developing exceptional products for the life sciences industry. We offer full-scale Custom Sugar-nucleotides Synthesis services. We are the preferred supplier for customers worldwide. Please feel free to contact us for more information about our Oligonucleotide-based Linker Modification Service.