The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.
The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.
5'-Nucleoside triphosphates ((d)NTPs) represent a class of vital biomolecules that are involved in countless processes and functions ranging from being the universal currency of energy to regulators of cell metabolism. In addition to their role in these fundamental biological transformations, their modified counterparts have advanced as a versatile and mild platform for the introduction of functional groups into oligonucleotides, a methodology that nicely complements the automated solid-phase synthesis that is usually applied1,2. Indeed, provided the (d)NTPs can act as substrates for RNA and DNA polymerases3, a wealth of functional groups including amino acids4-13, boronic acids14,15, nornbornene16, diamondoid-like residues17, side-chains for organocatalysis18, bile acids19, and even oligonucleotides20 can be introduced into oligonucleotides.
Beyond representing a convenient vector for the functionalization of nucleic acids, modified dNTPs can be engaged in SELEX and other related combinatorial methods of in vitro selection for the generation of modified catalytic nucleic acids21-30 and aptamers for various practical applications10,31-36. The additional side-chains that are introduced by the polymerization of the modified dNTPs are thought to increase the chemical space that can be explored during a selection experiment and supplement the rather poor functional arsenal of nucleic acids37. However, despite these attractive traits and the recent progress made in the development of both synthetic and analytical methods, no universally applicable and high-yielding procedure exists for the crafting of modified nucleoside triphosphates2,38.
The aim of this present protocol is to shed light into the (sometimes) intricate procedures leading to the synthesis and biochemical characterization of these activated building blocks (Figure 1B). Special emphasis will be given on all the synthetic details that often are difficult to find or are absent in experimental sections but are yet crucial for the successful completion of the synthetic pathway leading to the isolation of pure (d)NTPs (Figure 1).
1. Synthesis of the Modified Nucleoside Triphosphates
The synthetic approach chosen follows the procedure developed by Ludwig and Eckstein since this method is generally reliable and leads to very few side-products (Figure 1A)39.
2. HPLC Purification of the Modified Nucleoside Triphosphates
3. Primer Extension Reactions and TdT Polymerization
4. PCR with Modified Nucleoside Triphosphates
Modified nucleoside triphosphates are alluring synthetic targets since they allow for the facile introduction of an vast array of functional groups into nucleic acids41. However, the isolation and characterization of these activated building blocks is often revealed to be arduous. Consequently, the results shown herein are thought to provide a helping hand to follow the various steps within the aforementioned synthetic and biochemical procedures (Figure 1B).
In particular, Figure 3 shows a typical crude 31P-NMR spectrum of a modified dNTP (in this particular case, dUBpuTP (4)18, Figure 2), where the characteristic signals of the phosphorous centers can be observed (i.e. a doublet at -5.02 (Pγ), a doublet at -10.44 (Pα), and a triplet at -20.55 (Pβ) ppm). In addition, signals stemming for the diphosphate (two doublets at -4.84 and -10.63 ppm) and monophosphate (singlet at -0.18 ppm) side-products along with higher phosphates (signals at -21.02 and -23.19 ppm) are always observed at this stage. A first RP-HPLC analysis of the crude mixture is shown on Figure 4 (for dUBpuTP (4)) where the main peak (Rt = 30.78 min) corresponds to the 5'-triphosphate, while the main byproduct, the 5'-diphosphate, displays a slightly lower retention time (Rt = 30.03 min). Finally, after a thorough RP-HPLC purification, the modified dNTP needs to be characterized by NMR and MALDI-TOF (Figure 5). Both the 1H-NMR and 31P-NMR spectra are crucial for assessing the purity of the modified dNTPs, since the presence of undesired di- and mono-phosphates gives distinctive signals.
After establishing the purity of the nucleoside analogue and assessing the concentration of the stock solution either by mass or by UV-spectroscopy, the modified dNTP can be used in primer extension reactions in order to assess its substrate acceptance capacity by various polymerases. Figure 6 illustrates the outcome of primer extension reactions with dUtPTP (2), dAHsTP (6), and dCValTP (7) used either as lone modifications (lanes 5-7), as combinations of two modified dNTPs (lanes 8-10), or together along with the lone natural dGTP (lane 11).
Finally, Figure 7 shows representative TdT-mediated polymerization reactions with different modified dUTP analogues. In this context, dUcPTP (1) and dUFPTP (3) are the best substrates for TdT (lanes 1 and 4) since the tailing efficiencies are comparable or exceed those of the natural dTTP (lane 6). Instead, dUBpuTP (4) (lane 5) is a rather poor substrate in this context since little polydisperse longer-sized oligonucleotides can be observed.
Figure 1. A) Ludwig-Eckstein approach for the synthesis of (base) modified nucleoside triphosphates39. B) Schematic representation of all the steps required for the synthesis and biochemical characterization of modified dNTPs prior to their use in applications such as SELEX. Click here to view larger image.
Figure 2. Chemical structures of the modified nucleoside triphosphates: dUcPTP (1), dUtPTP (2), dUFPTP (3), dUBpuTP (4), dUBsTP (5),18 dAHsTP (6), and dCValTP (7) 13. Click here to view larger image.
Figure 3. 31P-NMR spectrum (121.4 MHz, D2O) of the crude reaction mixture of dUBpuTP (4). Click here to view larger image.
Figure 4. RP-HPLC profile of crude dUBpuTP (4): 0-100% eluent B in 40 min, flow rate: 3.5 ml/min (eluent A: 50 mM TEAB in H2O; eluent B: 50 mM TEAB in H2O/CH3CN (1/1)). Click here to view larger image.
Figure 5. Characterization of the modified nucleoside triphosphate dUBsTP (5): A) 31P-NMR spectrum (121.4 MHz, D2O, 128 scans);18 B) 1H-NMR spectrum (300 MHz, D2O, 128 scans); C) MALDI-TOF spectrum. Click here to view larger image.
Figure 6. Representative gel image (PAGE 15%) of primer extension reactions with various base modified dNTP analogues. Lane 1: primer; lane 2: natural dNTPs without dUTP; lane 3: natural dNTPs without dATP; lane 4: natural dNTPs without dCTP; lane 5: dUtPTP (2); lane 6: dAHsTP (6); lane 7: dCValTP (7); lane 8: dAHsTP (6) and dUtPTP (2); lane 9: dAHsTP (6) and dCValTP (7); lane 10: dUtPTP (2) and dCValTP (7); lane 11: dUtPTP (2), dAHsTP (6), and dCValTP (7); lane 12: natural dNTPs. Click here to view larger image.
Figure 7. Gel image (PAGE 20%) of the TdT polymerization reactions with various base modified dUTP analogues. Lane 1: dUcPTP (1); lane 2: dUtPTP (2); lane 3: dUBsTP (5); lane 4: dUFPTP (3); lane 5: dUBpuTP (4); lane 6: dTTP; lane 7: primer. Concentrations: 10 µM, 25 µM, 50 µM, 75 µM, and 100 µM. Click here to view larger image.
The inclusion of modifications into nucleic acids is of interest for numerous practical applications including the development of antisense and antigene agents42,43, labeling and functional tagging of oligonucleotides41, and in efforts to expand the genetic alphabet44-46. Chemical alterations and functional groups are usually introduced into nucleic acids by application of standard and automated solid-phase synthesis protocols. However, the phosphoramidite building blocks need to be resilient to the rather harsh conditions imposed by this methodology, which in turn imposes a severe restriction onto the nature of the chemical functionality47. Instead, the enzyme-mediated polymerization of modified nucleoside triphosphates allows for the introduction of a broader range of functionalities, since the sole restriction is that they act as substrates for polymerases1,2. Even though there is a noticeable lack of generally applicable methodology, reliable and robust synthetic and analytical methodologies have been developed for the synthesis of modified dNTPs. Moreover, due to their inherent nature, modified triphosphates are rather sensitive to different external conditions (e.g. pH, temperature) and thus, a detailed protocol for their synthesis and characterization is highly beneficial.
The workflow presented herein includes the chemical synthesis of modified dNTPs, RP-HPLC purification, NMR analysis, and enzymatic assays for the biochemical characterization of these nucleoside analogues. The most critical steps for a successful synthesis and characterization of modified dNTPs are the analysis of the crude product (by NMR), the thorough HPLC purification, the analysis of the purified material, and choice of an appropriate DNA (RNA) polymerase.
For the synthesis of the modified dNTPs we applied the method developed by Ludwig and Eckstein39, since fewer by-products are formed as compared to other procedures, albeit at the expense of a slightly longer synthetic route. Furthermore, the RP-HPLC purification certainly represents the pivotal step of the entire synthetic route since it will allow for the separation of the nucleoside diphosphates (dNDPs) which often strongly inhibit DNA and RNA polymerases48. After achieving the synthesis and purification, the purity of the resulting triphosphate needs to be assessed both by NMR and MALDI-TOF to ensure that no polymerase-inhibiting diphosphate is present.
The incorporation assay shown in Figure 6 clearly underscores the usefulness of this approach. Indeed, all of the dNTPs employed in this representative example reveal to be good substrates for the DNA polymerase (in this particular case Vent(exo–)) since no faster running bands corresponding to smaller fragments could be observed. Besides, the enzyme tolerates two substrates adorned with carboxylic acid residues (dUtPTP (3) and dCValTP (7)) and the polymerization of both dNTPs results in an oligonucleotide bearing not less than 39 additional negative charges. Another striking feature is that the bands resulting from the incorporation of modified dNTPs often display slower electrophoretic mobilities than the natural controls (compare e.g. lanes 11 and 12). Thus, primer extension reactions represent a powerful and yet simple way to assess the substrate acceptance of modified dNTPs.
Moreover, the TdT-mediated polymerization of triphosphates on the 3'-termini of single stranded oligonucleotides is an alluring strategy for the generation of highly functionalized nucleic acids49-52. The representative example shown in Figure 7 clearly demonstrates the procedure for selecting the functionalities that are tolerated by the Co2+-dependent TdT53. Indeed, dUcPTP (1) and dUFPTP (3), which are equipped with the proteinogenic amino acid L-proline and the dipeptide α-Phe-Pro, respectively, are the best substrates for the TdT and gave rise to tailing efficiencies that compare favorably to the unmodified dTTP control, even at concentrations as low as 10 µM (lanes 1 and 4). Surprisingly, analogue dUtPTP (2) where the proline residue is connected to the linker arm in trans compared to the free carboxylic acid, is not as good a substrate as its cis counterpart since polydisperse longer-sized oligonucleotides are only observed at higher dNTP concentrations (> 100 µM, lane 2). Furthermore, the sulfonamide-modified dNTP 5 is a moderate substrate for TdT and comparable to dUtPTP (2) (lane 3). In addition, dUBpuTP (4), which bears a strong hydrogen-bond donating motif, is a rather poor substrate for TdT and the polymerization reaction seems to be indifferent to the concentration of the modified dNTP. Thus, the following order of substrate acceptance can be construed from Figure 7: dUcPTP (1), dUFPTP (3)> dUtPTP (2), dUBsTP (5) > dUBpuTP (4).
Finally, the procedure described for polymerization reactions under PCR conditions using the modified analogues is rather similar to that involving natural dNTPs. However, the compatibility of modified dNTPs with polymerases under these conditions is of crucial importance for the generation of high-density functionalized nucleic acids7, especially with regards to their use in in vitro selection experiments.
In summary, the method for the synthesis and characterization of modified nucleoside triphosphates was emphasized and the establishment of such a protocol will certainly help in the development and crafting of novel analogues. Concomitantly, the emergence of such novel dNTPs will facilitate the generation of functionalized oligonucleotides; particularly, catalytic nucleic acids.
The authors have nothing to disclose.
This work was supported by the Swiss National Science Foundation (Grants n° PZ00P2_126430/1 and PZ00P2_144595). Prof. C. Leumann is gratefully acknowledged for providing the lab space and equipment, as well as for his constant support. Ms. Sue Knecht is acknowledged for fruitful discussions.
tributylammonium pyrophosphate | Sigma Aldrich | P8533 | Hygroscopic solid, keep under Ar |
2-chloro-1,3,2-benzodioxaphosphorin-4-one | Sigma Aldrich | 324124 | Moisture sensitive |
Pyridine | Sigma Aldrich | 82704 | Under molecular sieves |
Dioxane | Sigma Aldrich | 296309 | Under molecular sieves |
dimethylformamide (DMF) | Sigma Aldrich | 40248 | Under molecular sieves |
Acetonitrile | Fisher Scientific | HPLC grade | |
Triethylamine | Sigma Aldrich | 90342 | |
Tributylamine | Sigma Aldrich | 90781 | |
ddH2O | Milli-Q | deionized and purified water, autoclaved in the presence of Diethylpyrocarbonate (DEPC) | |
Diethylpyrocarbonate (DEPC) | Sigma Aldrich | 159220 | |
D2O | Cambridge Isotope Laboratories, Inc. | DLM-4-25 | |
Biochemical reagents | |||
g-[32P]-ATP | Hartmann Analytics | FP-301 | |
Natural dNTPs | Promega | U1420 | |
Vent (exo–) DNA polymerase | NEB | M0257S | |
DNA polymerase I, Large (Klenow) Fragment | NEB | MO210S | |
9°Nm DNA polymerase | NEB | MO260S | |
Terminal deoxynucleotidyl Transferase (TdT) | Promega | M828A | |
Pwo DNA polymerase | Peqlab | 01 01 5010 | |
T4 PNK | Thermo Scientific | EK0032 | |
Acrylamide/bisacrylamide (19:1, 40%) | Serva | 10679.01 | |
Agarose | Apollo Scientific | BIA1177 | |
G10 Sephadex | Sigma | G10120 | |
Urea | Apollo Scientific | BIU4110 | |
Equipment | |||
Jupiter semi-preparative RP-HPLC column (5m C18 300Å) | Phenomenex | ||
Gene Q Thermal Cycler | Bioconcept | BYQ6042E | |
PCR vials | Bioconcept | 3220-00 | |
HPLC system | Amersham Pharmacia Biotech | Äkta basic 10/100 | |
Oligonucleotides | |||
All oligonucleotides were purchased from Microsynth and purified by PAGE | |||
5'-CAAGGACAAAATACCTGTATTCCTT P1 | |||
5'-GACATCATGAGAGACATCGCCTCTGGGCTAAT-AGGACTACTTCTAATCTGTAAGAGCAGATCCCTGG-ACAGGCAAGGAATACAGGTATTTTGTCCTTG T1 | |||
5'-GAATTCGATATCAAG P2 | |||
More information on experimental procedures and equipment can be found in the following articles: | |||
Chem. Eur. J. 2012, 18, 13320 – 13330 | |||
Org. Biomol. Chem. 2013, DOI: 10.1039/C3OB40842F. |