Patent Application
20130143276
The increasing demand for 5′-adenylated DNA linkers and adapters correlates with the rapid progress in high throughput Next Generation Sequencing (NGS) of small RNAs. The construction of cDNA libraries for NGS requires attachment of 5′ and 3′ sequencing platform specific oligonucleotide adapters for downstream amplification, attachment that is facilitated by use of 5′-adenylated oligonucleotide linkers and adapters.
For first strand cDNA synthesis, an oligonucleotide adapter is ligated to 3′-end of an RNA polynucleotide. Annealing an oligonucleotide complementary to this attached adapter provides a priming site for copying the target RNA by reverse transcriptase. This protocol requires dephosphorylation of the RNA prior to ligation to prevent self-circularization or concatamerization of the RNA substrate. Self-ligation of the 3′ oligonucleotide adapter is also blocked, in this case by modification at the 3′-terminus. When subsequent steps require ligation of an oligonucleotide to the 5′ end of the RNA polynucleotide, for example to provide a second priming site for amplification of the reverse transcriptase cDNA product, the RNA must be re-phosphorylated to allow adapter ligation.
Use of adenylated linkers under conditions in which adenosine monophosphate (AMP) is not transferred to nucleic acids removes the need to dephosphorylate RNA substrates prior to ligation and prevents unwanted ligation products. Pre-adenylated oligonucleotide 3′ adapters may be used as a substrate in a ligation reaction with no ATP and either T4 RNA Ligase 1 (T4 Rnl1) (Lau, et al. Science 294(5543): 858-862 (2001)) or truncated version of T4 RNA Ligase 2 (T4 Rnl2) (Hafner, et al. Methods 44(1): 3-12 (2008)). This approach requires the use of an adenylated oligonucleotide adapter (AppDNA) as the ligation donor. Current methods for synthesis of AppDNA include either chemical synthesis or enzymatic synthesis.
A commonly used chemical method for pre-adenylation involves coupling of adenosine 5′-phosphorimidazolidate to 5′-phosphorylated oligonucleotide in solution or during solid phase oligonucleotide synthesis (Pfeffer, et al., Curr Protoc Mol Biol Chapter 26, Unit 26.4, John Wiley & Sons, Inc.: Hoboken, N.J. (2005)); Dai, et al., Org Lett 11 (5): 1067-1070 (2009)). This chemical method does not result in quantitative conversion of phosphorylated substrate to adenylated product. Consequently, purification is required to separate two closely related DNAs differing by a single nucleotide, further reducing the yield of desired product, and increasing the overall time and expense required to produce adenylated oligonucleotides.
A commonly used enzymatic method relies on T4 DNA ligase, requiring a multi-step process to create adenylated single-stranded DNA linkers. Since this enzyme requires a double-stranded DNA substrate, the single-stranded DNA linker is first annealed to an appropriately fashioned complementary oligonucleotide, then treated with T4 DNA ligase in the presence of ATP to adenylate the linker, and finally purified from the complementary DNA (Chiuman et al, Bioorg Chem 30 (5): 332-349 (2002); Vigneault et al., Nat Methods 5 (9): 777-779 (2008); Patel et al., Bioorg Chem 36 (2): 46-56 (2008); and U.S. published application 2010/0062494). As with the chemical synthesis, the multi-step aspects of this process increase both the time and expense required for production of the adenylated product.
In an embodiment of the invention, a method is provided for generating an adenylated oligonucleotide preparation that includes providing oligonucleotides having a 5′ phosphate; reacting the oligonucleotide with an ATP-sensitive ligase in the presence of an effective amount of ATP; and obtaining a stable reaction product in which greater than 70% or 80% or 90% of the oligonucleotides are adenylated. The effective amount of ATP is sufficient to permit adenylation while at the same time inhibit circularization of single-stranded DNA. For example, the effective amount may be in the range of 5 μM-10 mM ATP.
In an embodiment of the invention, the ATP-sensitive ligase is an RNA ligase and is thermostable, such as Methanobacterium thermoautotrophicum RNA ligase (MthRnl).
Examples of ATP-sensitive ligases for use in embodiments of the method include ligases with at least 90% sequence similarity with one or more of the ligases obtained from: Methanobacterium thermoautotrophicum; Pyrococcus abyssii; phage KVP40; Deinococcus radiodurans; Autographica California; Rhodothermus marinus; and phage TS2126.
Other examples of ATP-sensitive ligases include ligases having at least 90% amino acid sequence similarity to SEQ ID NO:1 or SEQ ID NO: 2.
In another embodiment, the method may, in addition to generating adenylated oligonucleotides, include ligating these adenylated oligonucleotides to polynucleotides by means of a second ligase such as a T4 RNA ligase, or mutants thereof such as a truncated T4 RNA ligase.
In an embodiment of the invention, the oligonucleotide may have either a blocked 3′ end or a free hydroxyl group at the 3′ end. The adenylation may be performed at a temperature in the range of 37° C.-70° C. and at a temperature-adjusted pH range of 5.5-8.0 (at 25° C.).
The gels in
It was found that the use of certain ATP-dependent ligases, here referred to as ATP-sensitive ligases, is an efficient, low-cost solution for obtaining 5′-adenylated oligonucleotides. These ligases can be used in an improved high-yield method to generate adenylated oligonucleotides suitable as linkers, as compared with existing methods employing chemical synthesis or T4 DNA ligase. The high yield of adenylated oligonucleotide in the absence of ligation products obviates the need for gel purification to remove a template strand or incompletely modified substrates, thus reducing the cost of synthesis.
The term “ATP-sensitive ligase” as used herein refers to an ATP-dependent ligase, more particularly ATP-dependent RNA ligase, that can efficiently generate adenylated oligonucleotides at ATP concentrations where ligation and circularization of the oligonucleotide is minimal as determined by gel electrophoresis.
The term “oligonucleotide” as used herein refers to a single-stranded DNA.
The term “stable reaction product” as used herein refers to the ability of a ligase reaction product to be sufficiently stable as to be capable of visualization by gel electrophoresis after removal from a reaction vessel.
Embodiments of the method allow quantitative conversion of 5′-phosphorylated oligonucleotides to the adenylated form and do not require addition of a template strand for adenylation to occur. The high yields simplify isolation and purification of the adenylated product. The characteristics of ATP-sensitive ligases, under conditions of increased ATP concentrations, enable high efficiency adenylation of substrates (including substrates with 3′ unprotected ends) in the absence of ligation.
ATP-sensitive RNA ligases for use in the present embodiments may include: MthRnl (optimum 60-65° C.) (Torchia, et al. Nucleic Acids Res 36(19): 6218-6227 (2008)); Pyrococcus abyssii (PAB 1020) containing an archael (thermostable) RNA ligase; phage KVP40 RNA ligase (Yin et al. Virology 319: 141-151 (2004)); Deinococcus radiodurans RNA ligase (Raymond et al. Nucleic Acids Res 35: 839-849 (2007)); Autographica California RNA ligase (Martins et al. J. Biol. Chem. 279 (18): 18220-18231 (2004)); Rhodothermus marinus RNA ligase (Blondel et al. Nucleic Acids Res 31: 7247-7254 (2003)); and phage TS2126 containing CircLigase™. Other suitable ATP-sensitive ligases include recombinant enzymes derived from the above-cited host cells and mutants thereof. Related ATP-sensitive ligases may be identified by BLAST searches using the amino acid sequence of any known ATP-sensitive ligase or derivative thereof and used to adenylate oligonucleotides.
In an embodiment of the method, adenylated oligonucleotides may be produced in a reaction that includes an ATP-sensitive ligase and amounts of ATP of at least 5 μM ATP, for example at least 10 μM ATP, for example at least 20 μM ATP, for example at least 50 μM ATP, for example at least 75 μM ATP, for example at least 100 μM ATP and as much as 500 μM ATP, for example 750 μM, for example 1 mM ATP, for example 10 mM ATP.
In an embodiment of the method, a thermostable RNA ligase may be used such as an Mth RNA ligase for adenylating oligonucleotides at a temperature of reaction in the range of 37°-70° C., for example 55°-65° C.
In an embodiment of the method, the reaction buffer contains Mg+2 or Mn+2 as a cofactor.
In an embodiment of the method, a suitable pH for an ATP-sensitive ligase-mediated adenylation of an oligonucleotide varies according to the buffer and the temperature of the reaction. For example, in the presence of Mg ions, a pH may be used in the range of pH 5.5-8.0, for example pH 6.5-7.0; whereas in the presence of manganese, a pH may preferably be used in the range of 5.0-7.0, for example pH 5.5-6.0 (adjusted at 25° C.).
In an embodiment of the method, the reaction may be incubated for as long as 5 hours or more, or as short as 5 minutes, to obtain an adenylated oligonucleotide product. The incubation time appears to be approximately inversely correlated to the amount of the ATP-sensitive ligase in the reaction mixture.
The above-described ATP-sensitive ligases can be distinguished from a second group of ligases characterized by a T4 RNA ligase that does not efficiently adenylate oligonucleotides under conditions that disfavor ligation. Embodiments of the method illustrate how an ATP-sensitive ligase can be screened for its ability to adenylate an oligonucleotide where the adenylated product is suitable for ligation to another oligonucleotide using a ligase of the type exemplified by T4 RNA ligase. For example, T4 Rnl2tr is unable to transfer AMP to the 5′ phosphate of a nucleic acid, and thus is only capable of catalyzing ligation if the RNA or DNA is already adenylated. The use of adenylated oligonucleotides with T4 Rnl2tr allows selective ligation of DNA primers to RNA for cloning or sequencing (Ho, et al. Structure 12(2): 327-339 (2004); Nandakumar and Shuman, Molecular Cell 16(2): 211-21 (2004)).
With the goal of efficient synthesis of 5′-adenylated oligonucleotides, a simple one-step protocol using an ATP-sensitive ligase has been developed. Optimization of conditions for this one-step protocol can be determined by a person of ordinary skill in the art without undue experimentation using embodiments of the method and assays described in the examples for CircLigase™ and MthRnl. ATP-sensitive ligases from sources other than those described in the examples may be identified and their oligonucleotide adenylation activity optimized by substitution of the ATP-sensitive ligase in the screening assays described herein with the test ligase. Conditions for optimization may include pH, temperature, amount of ATP, ratio of substrate to enzyme, and salt type and concentration.
Under optimized conditions, the yield of adenylated oligonucleotides can be as much as 70%, 80%, 90%, 95% or 98% using ATP-sensitive ligases. The high yield eliminates the need for additional purification from unadenylated forms. However, if purification is desired, then the ATP-sensitive ligase can be removed, for example by heat-killing followed by a proteinase K digestion, extraction with phenol-chloroform-isoamyl alcohol and removal by HPLC, thus providing a purified linker population that contains 5′ App. The recovery of adenylated polynucleotide linkers may be as much as 70%, 80%, 90%, 95% or 98% of the starting amount of polynucleotide.
Some advantages of using a thermostable ATP-sensitive ligase include: (a) the enzyme can be purified in high yields from an overexpressing strain of E. coli; (b) oligonucleotides can be adenylated without the need for adding a complementary strand; and (c) secondary structures in the oligonucleotides are reduced at elevated temperatures, and have a lower potential to interfere with adenylation of the 5′ end of the oligonucleotide.
All references cited herein, as well as U.S. provisional application Ser. Nos. 61/320,203 filed Apr. 1, 2010, 61/427,179 filed Dec. 25, 2010 and 61/427,178 filed Dec. 25, 2010, are hereby incorporated by reference.
A simple one-step protocol for screening for ATP-sensitive
RNA ligases capable of efficiently synthesizing 5′-adenylated oligonucleotides is provided. Initial screening of commercially available RNA ligases (T4 Rnl1, T4 Rnl2, CircLigase™ and MthRnl) showed that all ligases tested were capable for adenylating DNA to some extent although MthRnl produced adenylated product with the highest yield (
Reactions were performed using conditions recommended by the manufacturer. In general, reactions were carried out with an equimolar or lesser ratio of substrate to enzyme (S/E). Although MthRnl is known to form a homodimer, the monomer molecular weight was used for calculation of substrate/enzyme molar ratio in the reaction (
MthRnl, T4 Rnl1, T4 Rnl2, and T4 Rnl2tr were obtained from NEB, Ipswich, Mass. CircLigase™ was obtained from Epicentre Biotechnologies, Madison, Wis. Oligonucleotides used in this study were synthesized at Integrated DNA Technologies, Coralville, Iowa.
Oligonucleotides used in adenylation experiments:
RNA acceptors used in ligation experiments:
The histidine-tagged MthRnl was expressed in E. coli using a codon-optimized gene and the T7 expression system, and purified according to Torchia et al. (Nucleic Acids Res. 36: 6218-6227 (2008)).
During purification, mass spectrometry was used to assess whether column fractions contained the adenylated or free form of MthRnl. Based on this assay, column fractions were pooled to yield enzyme predominantly in the adenylated or free form.
Standard oligonucleotide adenylation reactions were performed in reaction mixtures (10 μl total) containing 50 mM sodium acetate, pH 6.0 buffer, 5 pmol of 3′-blocked, 5′-phosphorylated oligonucleotide, 100 μM ATP, 10 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 5 pmol (230 ng) of MthRnl (monomer). Assays were performed at 65° C. for 60 min, followed by inactivation of the enzyme by heating at 85° C. for 5 min. After addition of 5 μl formamide loading buffer, the reaction mixture was separated on a 15% Urea-TBE denaturing polyacrylamide minigels (Invitrogen, now Life Technologies, Carlsbad, Calif.), stained with SYBR® Gold (Invitrogen, now Life Technologies, Carlsbad, Calif.) and visualized using an AlphaImager HP (Alpha Innotech, now Cell Biosciences, Santa Clara, Calif.).
The DNA circularization assay was performed using an excess of pre-adenylated MthRnl (25 pmol monomer), and 5 pmol of 3′-OH, 5′-phosphorylated oligonucleotide, and variable concentrations of ATP. The circularity of ligated DNAs was identified via resistance to Exonuclease I (NEB, Ipswich, Mass.) digestion. The presence of adenylated DNA was confirmed by ESI-MS analysis (
For preparative DNA adenylation, 300 μM of DNA substrate and 150-300 μM of enzyme monomer were used in reactions as described above in appropriately scaled reaction volumes at 65° C. for 2 hours, and the extent of reaction assessed on 5 pmol DNA aliquots by gel electrophoresis. The remainder of the reactions was heat-inactivated at 85° C. for 5 min, treated with Proteinase K (NEB, Ipswich, Mass.), extracted with phenol-chloroform-isopropanol (25:24:1), chloroform-isopropanol (49:1) and ethanol-precipitated.
MthRnl was incubated in a 10 μl reaction with an oligonucleotide, having a 5′ phosphate and a 3′ NH2. The reaction contained: 1 μl 10× reaction buffer, 10 μmol of substrate, 1.25-10 μmol of MthRnl (460 ng of ligase=10 pmole of monomer), and 100 μM ATP. The 10× reaction buffer contained 0.50 M NaOAc, pH 6.0, 50 mM DTT, 1 mM EDTA and 0.10 M MgCl2.
Assays were performed at 65° C. for 60 minutes. The enzyme was inactivated by heating at 85° C. for 5 min. After the addition of 5 μl formamide, reaction products were separated on 15% Urea-TBE denaturing minigels (Invitrogen, now Life Technologies, Carlsbad, Calif.), stained with SYBR® Gold (Invitrogen) and photographed using AlphaImager HP (Alpha Innotech, now Cell Biosciences, Santa Clara, Calif.). The results are shown in
Further optimization of the DNA adenylation reaction was performed for MthRnl, although it is envisaged that the optimized protocol described herein is suited to the entire class of ATP-sensitive RNA ligases described herein. A person of ordinary skill in the art may make minor adjustments with respect to temperature optima according to the thermostability of the RNA ligase.
MthRnl was incubated with a 50 nt oligonucleotide with a 5′ phosphate and a 3′ OH, and 17 nt oligonucleotide with 5′ phosphate and 3′ amino block in a 10 μl reaction. The reaction contained: 1 μl 10× reaction buffer, 5 pmol of substrate, and 5 or 10 pmol of MthRnl (460 ng of ligase=10 pmole of monomer). The 10× reaction buffer contained 0.50 M NaOAc, pH 6.0, 50 mM DTT, 1 mM EDTA and 0.10 M MgCl2. The temperature and time for the assays were the same as Example 1. The reactions contained variable amounts of ATP. The Mth ligase reactions with about 50 μM ATP resulted in enhanced adenylation. An amount of about 500 μM ATP blocked self-ligation of DNA molecules that have a free 3′ OH. The results are shown in
When the concentration of ATP was increased, two bands were observed, where one product ran one nucleotide slower than substrate on denaturing polyacrylamide gel and corresponded to AppDNA. Synthesis of AppDNA was confirmed by functional analysis (
High ATP concentrations (greater than 5 μM ATP) inhibited DNA circularization with pre-adenylated MthRnl as shown in
For MthRnl, ATP is an effective inhibitor of the DNA ligation step. When the concentration of ATP was increased, a product accumulated that ran one nucleotide slower than substrate on denaturing polyacrylamide gel, and presumably is AppDNA (see
The DNA oligonucleotide adenylated by MthRnl (as described in Example 4) was ligated to RNA by T4 Rnl2tr. 10 μl ligation reactions containing 5 pmol of the RNA acceptor (SEQ ID NO: 11 or SEQ ID NO:12), 7 pmol adenylated pDNA17c-NH2 in 10 mM Tris-HCl pH 7.5 buffer, 10 mM Mg, 1 mM DTT and 200 U of T4 Rnl2tr were incubated for 2 hours at 25° C. Reactions were stopped by adding 5 μl formamide loading buffer, heat-inactivated at 85° C. for 5 minutes. Ligation reactions were separated on 15% TBE urea containing polyacrylamide gels, and products were visualized by staining with SYBR® Gold (Invitrogen, now Life Technologies, Carlsbad, Calif.) and scanning on a GE Healthcare Life Sciences (Piscataway, N.J.) Typhoon 9400 variable mode imager.
Ligated products were observed in the reaction that contained the MthRnl-adenylated oligonucleotide, RNA and T4 Rnl2tr (
A 5′ phosphorylated 21 nt oligonucleotide, 5′ pTCG TAT GCC GTC TTC TGC TTG-NH2 (SEQ ID NO:4) 3′ amino block, was reacted with MthRnl under the conditions described in Example 1 using 100 μM ATP. Reacted and unreacted oligonucleotides were analyzed by electrospray ionization mass spectrometry according to the method of Shah and Friedman (Nature Protocols 3(3): 351-6 (2008)). Samples (10 mM oligonucleotides in 50% acetonitrile, 1% triethylamine) were conducted by direct infusion (10 ml/min) into a 6210 ESI-TOF mass spectrometer with an electrospray ionization source (Agilent Technologies, Hollis, N.H.). Data were acquired in negative ion mode, from m/z 500 to 8000 (high mass range enabled). The VCap and Fragmentor values were set to 4000 and 215 V, respectively. The drying gas flow rate was 7 L/min, the gas temperature was 300° C. and the nebulizer was set to 25 psig. The results are shown in the top and bottom panels of
Gene databases can be interrogated using part or all of the sequences of the two ATP-sensitive RNA ligases defined by SEQ ID NO: 1 or SEQ ID NO: 2, or using sequences having at least 90% sequence homology with SEQ ID NO: 1 or 2. Candidate enzymes are defined by sequence matches that share at least 90% sequence identity with at least 10% or 20% of SEQ ID NO: 1 or 2. These candidate enzymes can be synthesized and assayed as described herein for MthRnl.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/30881 | 4/1/2011 | WO | 00 | 10/24/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61320203 | Apr 2010 | US | |
| 61427179 | Dec 2010 | US | |
| 61427178 | Dec 2010 | US |
