5’ Adenylation (5’ adenylate, 5’-adenylpyrophosphoryl or rApp)
Rapid progress has been made in recent years in high-throughput next-generation sequencing of small RNAs, which has increased the demand for 5’-adenylated DNA linkers or adapters. 5’ adenylation is an important biochemical step needed for the following applications:
- Automated library preparation,
- of 3-kb mate-pair libraries,
- Preparation of 8-kb mate-pair libraries for Illumina sequencing protocols,
- Automated index library preparation,
- Preparation of non-indexed libraries,
- Manual library preparations.
Libraries are needed for Next-generation sequencing technologies, however, at present, next-generation sequencing platforms use slightly different technologies for sequencing, such as pyrosequencing, sequencing by synthesis or sequencing by ligation. Most platforms use a common library preparation procedure, some of them with minor modifications, before a 'run' on the sequencing instrument is started. The major features of this procedure include fragmenting the DNA using sonication, nebulization or shearing, followed by DNA repair and end polishing of blunt ends or A overhangs and, finally, platform-specific adaptor ligation.
Linkers or adaptors are short single-stranded or double-stranded oligonucleotides that can be ligated to the ends of other DNA or RNA molecules. Linkers or adaptors can contain overhangs and no overhangs, but both can be ligated to PCR products and linearized plasmids. The use of adaptors allows the introduction of restriction enzyme sites, amino acid codons, and other desired sequence motifs to the selected DNA or RNA strands. Linkers or adaptors can be used for cloning libraries or specific genes, as well as the introducing of peptide tag sequences.
For the construction of cDNA libraries for next-generation sequencing technologies, the attachment of 5’ and 3’ sequencing platform specific adapters for downstream amplification is required. A 3’ adapter is ligated to the 3’-end of the RNA to introduce a sequence for annealing the primer used by reverse transcriptase for the first-strand cDNA synthesis. The dephosphorylation at the 5’-end of RNA is required prior to the ligation reaction to prevent self-circularization. A modification at the 3’-terminus of the DNA adapter with a blocking group, such as a 2’,3’-dideoxynucleotide or a 3’-propyl spacer, helps to prevent self-ligation. For the ligation of an adapter to the 5’-end of the RNA, prior to reverse transcription, the RNA must be re-phosphorylated to allow adapter ligation. However, cDNA libraries can be constructed using pre-adenylated single-stranded DNA (AppDNA) as a substrate in a 3’ adapter ligation reaction with no ATP and the help of either T4 RNA ligase 1 or a truncated version of T4 RNA ligase 2.
The use of
5’-adenylated linkers remove the need to dephosphorylate the RNA prior to ligation and prevents unwanted ligation products.
The following graphic shows the structure of a 5’-adenylated RNA (5’ AppRNA).
Structure of 5’-adenylated RNA (5’ AppRNA).
Pre-adenylated single-stranded DNA (AppDNA) is used for cDNA library construction as a substrate in a 3’ adapter ligation reaction with no ATP, and either T4 RNA ligase 1 or a truncated version of T4 RNA ligase 2 in improved protocols.
Ligation is a critical, cellular biochemical reaction that joins together nucleic acid segments. The ligation reaction requires an upstream acceptor nucleic acid sequence and a downstream nucleic acid sequence, as well as the enzyme ligase and all needed co-factors. In some cases, a template may also be needed.
DNA ligases together with RNA ligases and mRNA capping enzymes are part of the nucleotidyl transferase superfamily. Ligases interact with a nucleotide cofactor to form a covalent enzyme-nucleoside monophosphate. DNA ligases utilize either adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD+) as the nucleotide cofactor.
DNA Ligases
The enzyme DNA ligase forms two covalent phosphodiester bonds between the 3' hydroxyl end of one nucleotide and the 5' phosphate end of another. ATP is required for the three step ligation reaction:
| Step 1: |
A phosphoamide bond forms between the amino group of an active site lysine and the 5’ phosphate of AMP for DNA and RNA ligases, or GMP for mRNA capping enzymes. The activated enzyme-nucleotide monophosphate adduct or intermediate is generated in the absence of a nucleic acid substrate. Inorganic pyrophosphate is released in this step by enzymes utilizing nucleoside triphosphates, whereas nicotinamide mononucleotide is released by NAD+-dependent ligases. |
| Step 2: |
The 5’ phosphate group of nucleotide monophosphate is transferred from the active site lysine to a phosphorylated DNA 5’ end where it forms a pyrophosphate linkage (5’P-5’P). The 5’ adenosine mono phosphate activates the 5’ phosphate of a DNA substrate for phosphodiester bond formation. |
| Step 3: |
The 3’ hydroxyl of an adjacent DNA strand attacks the 5’ phosphorylated DNA end to displace adenosine mono phosphate and covalently join the DNA strands |
The following figure shows the structure model of a DNA ligase.
Structure model of a DNA ligase.
The helix-hairpin-helix (HhH) domain of Thermus filiformis(Tfi) DNA ligase is shown. The HhH domain of the NAD+-dependent DNA ligase from Thermus filiformis has four helix-hairpin-helix motifs arranged on one surface with a similar twofold symmetry. The nicked DNA from the DNA ligase I structure is positioned above these DNA-binding elements, indicating that they are properly spaced to bind to the minor groove.
DNA, with a 5’-adenylpyrophosphoryl cap (5’-adenylated DNA; AppDNA), is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5’-Adenylated DNA is also useful for in vitro selection experiments, therefore, an efficient preparation of 5’-adenylated DNA is desirable for several biochemical applications.
RNA Ligases
RNA ligases are involved in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. RNA ligases join 3’-OH and 5’-PO4 RNA termini through a series of three nucleotidyl transfer steps similar to DNA ligases.
| Step 1: |
RNA ligase reacts with adenosine triphosphate (ATP) to form a covalent ligase-(lysyl-N)-adenosine monophosphate (AMP) intermediate. Pyrophosphate is also released. |
| Step 2: |
AMP is transferred from ligase-adenylate to the 5’-PO4 RNA end. A RNA-adenylate intermediate (AppRNA) is formed. |
| Step 3: |
The 3’ hydroxyl of an adjacent RNA strand attacks the 5’ phosphorylated DNA end to displace and release adenosine mono phosphate and covalently join the RNA strands via a phosphodiester bond. |
The following shows the structure of T4 RNA ligase (2HVR).
RNA Ligase-Nucleic Acid Complex Structures.
Two views of the RNA ligase-nucleotide complex are illustrated. The adenylated 3’H/2’OH nick can be seen in the middle of the left structure where the two oligo stands that will be ligated meet (gray and yellow colored strands).
Mechanism of the three-step pathway of nick sealing by ATP-dependent polynucleotide ligases.
Step 1: : Attack of the active-site lysine on the α-phosphorus of ATP to form a covalent ligase-AMP intermediate by releasing pyrophosphate (PPi).
Step 2: Binding of ligase-AMP at the nick followed by AMP transfer to the nick 5’ phosphate to form a 5’ adenylated nicked duplex.
Step 3: : The nick 3’ OH group attacks the nick 5’ phosphate to form a phosphodiester bond and AMP is released.
Both types of DNA ligases, either utilizing ATP or NAD+ as cofactors, share basically the same catalytic mechanism to join the separate ssDNA strands together. The crystal structure of the
Tfi DNA ligase protein has a modular architecture and contains the following domains: an adenlylation domain, an oligonucleotide binding (OB-fold) domain, a Zinc finger motif domain, a helix-hairpin-helix (HhH) and a C-terminal domain of a breast cancer susceptibility protein BRCT domain (C-terminal domain of a breast cancer susceptibility protein). As can be seen in the next illustration the binding of ATP in the active site also requires magnesium ions.
Models of the active site (left) and reaction steps (right) for the
Tfiligase are shown. (See Lee et al. 2000 for more deatail). The residues that are thought to participate in binding of metal ionas and the 5’phsophate end of the nick are indicated. Different domains are color-coded. Adenylation; blue, OB domain; green, Zink-finger and HhH domain; orange, BRCT domain; gray, DNA; red, and AMP; cyan. A: The apo enzyme is elf-adenylated. B: A duplex DNA is bound to the ‘non-catalytic’ DNA-binding site. C:
TFI ligase slides along DNA and recognizes the nick. D: Duplex DNA is kinked at the nick and the kinked DNA is bound to both the ‘catalytic’ and ‘non-catalytic’ DNA-binding sites. E: The binding triggers a rearrangement of the protein domains. Note: magnesium ions are needed at this step. AMP is de-adenylated from lysine 116 and is transferred to the 5’-phosphate of the nicked site, and magnesium ions are bound. F: Nick closure occurs, the DNA strands are ligated, the ligated duplex DNA is detached from the ‘catalytic’ DNA-binding site, and the domain movements restore the ligase conformation during the release of the duplex DNA and to get ready for another reaction cycle.
RNA ligases are essential reagents for many methods in molecular biology, including next generation (NextGen) RNA sequencing. The enzymes T4 phage RNA ligases 1 and 2 are used for ligation of RNA and for rapid amplification of cDNA ends (RLM-RACE), 3’ RNA labeling, and sealing nicks in dsRNA and dsRNA-DNA hybrids. Phage TS2126 ligase is used for ssDNA circularization and the
Mth RNA ligase from thermophilic archaeal bacteria
Methanobacterium thermoautotrophicum is used for enzymatic synthesis of 5’ adenylated DNA linkers. Furthermore, RNA ligases can be used to construct cDNA libraries, which are needed for next generation sequencing of small RNAs or other applications.
Custom synthesis of 5' adenylated RNA or DNA
Bio-Synthesis offers oligo 5' adenylation starting 1.0 umole scale and up. 5' adenylated oligos require an extra purification after adenylation step adn are not compatible with heat-sensitive modificaitons. To prevent a 5' adenylated oligo from sel-ligating, a block group is required on teh 3'-end, such as a 2',3'-Dideoxynucleotide or 3' (C3) Propyl spacer.
Bio-Synthesis also offer other capped RNA
oligonucleotide synthesis. For more information on
Capped Oligonucleotide Synthesis
References
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Lee JY, Chang C, Song HK, Moon J, Yang JK, Kim HK, Kwon ST, Suh SW; Crystal structure of nad(+)-dependent DNA ligase: modular architecture and functional implications.
Embo J. (2000) 19 p.1119.
Nandakumar J, Shuman S, Lima CD; Rna ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward.
Cell(Cambridge,Mass.) (2006) 127 p.71
Nandakumar J, Ho CK, Lima CD, Shuman S.; RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J Biol Chem. 2004 Jul 23;279(30):31337-47. Epub 2004 Apr 13.
Maha P. Patel, Dana A. Baum, Scott K. Silverman; Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand. Bioorganic Chemistry 36 (2008) 46–56.
Sriskanda V, Shuman S.; Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase. Nucleic Acids Res. 2002 Feb 15;30(4):903-11.
YANGMING WANG and SCOTT K. SILVERMAN Efficient RNA 59-adenylation by T4 DNA ligase to facilitate practical applications. RNA (2006), 12:1142–1146.
Alexander M Zhelkovsky and Larry A McReynolds; Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase – engineering a thermostable ATP independent enzyme. BMC Molecular Biology 2012, 13:24.
Zhuang F, Fuchs RT, Sun Z, Zheng Y, Robb GB.; Structural bias in T4 RNA ligase-mediated 3'-adapter ligation. Nucleic Acids Res. 2012 Apr;40(7):e54. doi: 10.1093/nar/gkr1263. Epub 2012 Jan 12.
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