Phototriggered DNA Phosphoramidate Ligation in a Tandem 5

Sep 17, 2012 - Santa Fe Institute, 1399 Hyde Park Road, Santa Fe, New Mexico 87501, United States. Bioconjugate Chem. , 2012, 23 (10), pp 2014–2019...
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Phototriggered DNA Phosphoramidate Ligation in a Tandem 5′Amine Deprotection/3′-Imidazole Activated Phosphate Coupling Reaction Jonathan L. Cape,† Joseph B. Edson,† Liam P. Spencer,† Michael S. DeClue,† Hans-Joachim Ziock,‡ Sarah Maurer,‡,§ Steen Rasmussen,‡,∥ Pierre-Alain Monnard,‡,§ and James M. Boncella*,† †

Material, Physics and Applications Division and ‡Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States § Center for Fundamental Living Technology, Institute for Physics & Chemistry, University of Southern Denmark, DK-5230 Odense, Denmark ∥ Santa Fe Institute, 1399 Hyde Park Road, Santa Fe, New Mexico 87501, United States S Supporting Information *

ABSTRACT: We report the preparation and use of an Nmethyl picolinium carbamate protecting group for applications in a phototriggered nonenzymatic DNA phosphoramidate ligation reaction. Selective 5′-amino protection of a modified 13-mer oligonucleotide is achieved in aqueous solution by reaction with an N-methyl-4-picolinium carbonyl imidazole triflate protecting group precursor. Deprotection is carried out by photoinduced electron transfer from Ru(bpy)32+ using visible light photolysis and ascorbic acid as a sacrificial electron donor. Phototriggered 5′- amino oligonucleotide deprotection is used to initiate a nonenzymatic ligation of the 13-mer to an imidazole activated 3′-phospho-hairpin template to generate a ligated product with a phosphoramidate linkage. We demonstrate that this methodology offers a simple way to exert control over reaction initiation and rates in nonenzymatic DNA ligation for potential applications in the study of model protocellular systems and prebiotic nucleic acid synthesis.

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INTRODUCTION Template-directed synthesis of oligonucleotides by nonenzymatic methods is widely used to study mechanisms of prebiotic genetic replication1−6 and is playing an increasing role in synthetic biological systems.7 Typical methods of nonenzymatic, metal-ion catalyzed DNA and RNA synthesis use imidazole activated 3′ or 5′-phosphate nucleoside monomers and oligonucleotides in aqueous solution to achieve phosphodiester bond formation in template-directed primer extension8,9 reactions or oligonucleotide ligations.10−13 A steady stream of improvements in our understanding of imidazole activated phosphate synthesis,14−19 stability,20 and optimal reaction conditions1,7,21−29 has been reported since the first applications of activation procedures by Schneider-Bernloehr,30−32 Sulston,1,15 and Weimann2 over 40 years ago. Despite these improvements, several disadvantages have yet to be addressed, including the lack of exogenous control in reaction initiation, uncontrolled steady state rates, and low reaction yields due to competition between phosphodiester bond formation and the hydrolysis of imidazole activated monomers. This work presents a new protecting group strategy for 5′-amino oligonucleotides that addresses some of these limitations. This strategy uses a visible light triggered deprotection of an N© 2012 American Chemical Society

methyl picolinium carbamate protecting group to unmask the 5′-amino group of the oligonucleotide, which can then react with a hairpin that is activated on its 3′-end with an imidazolephosphate to yield a phosphoramidate linkage. Previous work toward 5′-photoactivated protecting groups have utilized 5′-dye carbonates including dimethoxybenzoincarbonates33,34 and α-methyl-2-nitropiperonyloxycarbonyl35−37 functionalities to protect 5′-hydroxyl groups. While these protecting groups exhibit fast deprotection kinetics, they require near UV-photolysis (95%) by treatment with an excess of the Nmethyl-4-picolinium carbonyl imidazole triflate protecting group precursor (4) in distilled water for 48 h in the dark at room temperature. HPLC purification and MALDI-TOF MS indicate that only a single N-methyl picolinium protecting group is attached to the oligonucleotide (m/z = 4626, see Supporting Information Figure S2). The protected oligonucleotide (2′) does not react with the 3′-phosphorimidazolide of (1), indicating that this protecting group blocks the reactivity of the 5′-amino group of (2). Deprotection of (2′) is achieved by photoinduced electron transfer from Ru(bpy)32+ using an excess of ascorbic acid as a sacrificial electron donor (Scheme 2). Reaction rates are controllable by Ru(bpy)32+ concentration (see Supporting Information Figure S4) and its excitation efficiency. Deprotection of (2′) is complete within 20 min under the following conditions: 25 μM Ru(bpy)32+, 10 μM oligonucleotide, 2 mM ascorbic acid, 100 mM HEPES buffer (pH 7.4), mono-

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RESULTS AND DISCUSSION This work describes the use of the N-methyl picolinium carbamate protecting group in a tandem photodeprotection/ ligation reaction. A 55-mer sequence (1), shown in Scheme 1, forms a 25-mer hairpin sequence along with a 30-mer 5′overhang section that serves as the template. The 55-mer is also terminated by a 3′-phosphate group which is activated with an imidazole leaving group. The 13-mer oligonucleotide (5′NH2TCCCGGGTTTTTT) (2) can hybridize with the overhang sequence of (1). Its 5′-amino group is initially derivatized with the picolinium carbamate protecting group, and hence can 2015

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Figure 1. Panel A: HPLC traces showing progression of the reaction at 25 °C of protected (2′) (10 μM) with the phosphorimidazolide derivative of (1) (10 μM) under continuous photolysis (400 nm) at t = 0, 5, 15, and 30 min. Arrows and labels above the traces indicate the conversion of protected (2′) to (2), consumption of 3′-imidazole activated 3′-phosphate derivative of (1), formation of ligated product (3), and the appearance of a photochemically decomposed form of (3) [(3)-decomp].

Figure 3. Apparent rate constants for consumption of reactants (1) and (2′) and the formation of products (3) and (3-decomp). Firstorder exponential fits to the data in Figure 2 were used estimate these rate constants (see Supporting Information, including Figure S8).

in the near UV (250−350 nm) and at its visible peak at 450− 500 nm. Changing the excitation wavelength to 550 nm, outside of the spectral extent of the Ru(bpy)32+ MLCT band, results in no conversion to the deprotected form after 30 min (see Supporting Information Figure S7). Under continuous photolysis, reaction of the deprotected 5′amino oligomer (2) with the 3′-imidazole activated 3′phosphate of (1) results in the formation of a 68-mer product

chromatic (400 nm) photolysis provided by the Xe-arc lamp and excitation monochromator of a Shimadzu RF1000 fluorimeter. This visible wavelength was chosen to avoid direct excitation and subsequent degradation of the 3′-FAM fluorescent tag on (2′), which has significant absorbance both

Figure 2. Reaction kinetics from HPLC analyses for tandem photochemical deprotection/nonenzymatic ligation performed at 5 °C (A), 25 °C (B), 45 °C (C), and 60 °C (D). All reactions go to completion relative to their limiting reactant and overall reaction rates increase continuously with temperature. Quantification of peak areas and data workup are in the Supporting Information. 2016

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reaction rates for reactant consumption and product formation throughout the entire temperature range suggest that intermediate steps between the photochemical deprotection and coupling steps are either comparable in rate or faster than the deprotection step, without the accumulation of intermediate species (such as deprotected (2)). Thus, at all temperatures hybridization and coupling of (1)/(2) is faster than photochemical deprotection. At higher temperatures, we hypothesize that coupling occurs through a transient hybridized complex with a lifetime that is longer than 1/kligation. This work establishes reaction conditions and a plausible mechanism for a model phototriggered DNA-ligation reaction. This reaction is demonstrated to have high efficiency and can be performed both above and below the Tm of the activated template/complement pair, but does require at least transient complexation of the template and compliment sequences for the reaction to occur. We anticipate that this protection/ deprotection method will greatly assist current efforts toward understanding self-assembly and compartmentation of model genetic materials in membranous cell-like systems.42 One challenge in this area is exerting control over reaction initiation and rates for nonenzymatic ligation reactions. The methodology described in this work should allow one to assemble and control ever more complex vesicle enclosed/compartmented systems by overcoming the difficulties of uncontrolled ligation of encapsulated genetic materials placed in these systems. Although these results do demonstrate some undesirable properties for phototriggered DNA-ligation, including the possible degradation of an end-capping fluorescein tag, these difficulties can likely be circumvented by the appropriate choice of model oligonucleotides and photolysis conditions. Work is currently underway to integrate the photochemical deprotection/ligation reactions described here with a second similar deprotection chemistry for lipid precursors;43 the goal of which is to generate a protocellular system which grows and replicates both its containing membrane system and its bound model genetic material.44,45

Scheme 3. Proposed Reaction Mechanism

(3) as shown by the chromatograms and kinetic profile in Figure 1. The ligation reaction is completed after 45 min, but the chromatograms indicate that a nonfluorescent degradation product ((3)-decomp) continues to accumulate at the expense of the ligated product (3). MALDI analyses of HPLC purified reaction product confirmed the identity of (3), and established that the byproduct was ∼314 m/z smaller than the initial ligated product (see Supporting Information Figure S1). The MS data and observation of nonfluorescence strongly suggest that (3)-decomp is formed from the degradation of the 3′-FAM tag. Control experiments that did not contain Ru(bpy)32+ exhibited no degradation of the 3′-FAM tag, indicating a photoredox mechanism involving the ruthenium photosensitizer. Further kinetic studies were undertaken to distinguish whether the deprotection/nonenzymatic coupling reaction occurs through a template directed mechanism, or through random reaction of nonhybridized (1) and (2). The rates of deprotection and coupling were ascertained at various temperatures above and below the expected Tm of the (1)/(2) complex. Figures 2 and 3 demonstrate that the rate for consumption of (1) and (2′) are similar to the rate of product (3) + (3-decomp) formation at each temperature. First-order exponential fits to the data (see for example Supporting Information Figure S8) give apparent rate constants for these reactions in Figure 3 and demonstrate that overall rates rise by a factor of 3.5 over the 5−60 °C temperature range. The continuous increase of the rate constants as a function of temperature indicates that melting of the (1)/(2) complex does not bring about rate limitation in this tandem reaction. Some accumulation of deprotected (2) is noted in the kinetic traces of Figure 2. This accumulation is due to the superstoichiometric initial concentration of (2′) relative to (1) in several of these reactions, and is not indicative of (2) being accumulated as a reaction intermediate if the ligation reaction were rate limiting. Additional experiments (see Supporting Information Figure S3) were conducted using a truncated sequence of (1) (i.e., (5)) for which nearly all of the base-pairing sequence with (2) was eliminated. Importantly, no ligated product is observed in identical reactions at 25 °C using the 3′-activated form of the truncated sequence (sequence (5) in Scheme 1 contains only a single A overhang available to base pair to the 5′-thymidine of (2′)). Here, we observed only deprotection of (2′) and accumulation of (2) with no subsequent formation of a ligated product. The following mechanistic conclusions can be drawn from these data and are illustrated in Scheme 3: (1) The absence of reaction of the truncated sequence unambiguously indicates the need for some form of hybridization with the template in the coupling reaction at room temperature, and (2) the similar

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ASSOCIATED CONTENT

S Supporting Information *

Material and methods, full synthetic procedures, and characterization data of the synthesized compounds are presented. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (505) 665-0795. Fax: (505) 667-9905. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for many fruitful discussions and suggestions from colleagues at Los Alamos National Laboratory and the University of Southern Denmark. Different aspects of this work were supported by the Laboratory-Directed Research and Development program at Los Alamos National Laboratory, in particular through the “Protocell Assembly” and “Coupling of genetics and metabolism and the origin of life” projects therein, as well as the NASA grant NNH08AI88I, the EC FP7 grant MatchIT (grant agreement no. 249032), and the Center 2017

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for Fundamental Living Technology (FLinT) at the University of Southern Denmark. We also thank the reviewers for their insightful comments that greatly improved this manuscript.

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dx.doi.org/10.1021/bc300093y | Bioconjugate Chem. 2012, 23, 2014−2019