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Photocleavable aminotag phosphoramidites for 5[prime]-termini DNA/RNA labeling
Introduction
Materials And Methods
Synthesis of photocleavable aminotag phosphoramidites
5[prime]-PC-amino-oligonucleotide synthesis
Reaction with activated marker molecules and photocleavage
Hybridization and colorimetric detection
Reaction of 5[prime]-PC-amino-oligonucleotide with activated support and photocleavage
Results
Design of photocleavable aminotag-phosphoramidites
Synthesis and evaluation of PC-amino-oligonucleotides and their conjugates
Evaluation of a PC-amino-oligonucleotide in hybridization assay
Reaction of PC-amino-oligonucleotides with activated supports and photocleavage-mediated affinity purification
Discussion
Acknowledgements
References
Photocleavable aminotag phosphoramidites for 5[prime]-termini DNA/RNA labeling
ABSTRACT
INTRODUCTION
The detection and isolation of biomolecules are two central themes in biotechnology. In the case of DNA/RNA analysis, important manipulations include the modification of DNA/RNA for detection as well as immobilization purposes. To date, numerous methods have been developed for the incorporation of ligands into synthetic oligonucleotides using phosphoramidite chemistry (1). One approach is based on the incorporation of an aliphatic amino group into synthetic oligonucleotides by use of amino-modifier phosphoramidites (2-4) or modified CPGs (5). Such amino groups allow the post-synthetic addition of a variety of molecules including fluorescent markers, biotin, hapten or enzyme which can be used as hybridization probes for non-radioactive detection of specific target DNA/RNA (6) or as sequencing primers (7). Amino-modified oligonucleotides have also been used for preparation of affinity matrices (8) and as immobilized PCR primers (9).
One disadvantage of covalent labeling and immobilization of oligonucleotides is that the covalent bond is not selectively cleavable, and therefore does not allow for the recovery/release of oligonucleotide or the removal of the label. In order to circumvent this problem, several approaches have been proposed based on chemical cleavage. These include the use of cleavable disulfide bonds (10,11) or the introduction of acid cleavable linkers (12,13). These approaches, however, require the application of chemical reagents that are not suitable for many applications. They also necessitate removal of the excess of cleaving reagents and result in residual modification of the oligonucleotide.
We describe here a new approach to manipulation of synthetic oligonucleotides based on the use of photocleavable aminotag phosphoramidites enabling specific cleavage of covalent bonds formed with 5[prime]-oligonucleotide amine groups. Photocleavable aminotag phosphoramidites (PC-aminotag) introduce a photocleavable aliphatic amino group onto the 5[prime]-terminus of synthetic oligonucleotides. These reagents represent a more general approach compared to the recently described PC-biotin phosphoramidite (14), which directly introduces a photocleavable biotin moiety onto the 5[prime]-end. In contrast, 5[prime]-PC-amino-oligonucleotides can be reacted with a wide variety of activated molecules and surfaces, thereby facilitating the formation of many different photocleavable conjugates. Upon exposure to near-UV light such photocleavable conjugates allow for the recovery/release of unmodified oligo-nucleotide and/or a marker molecule.
Two PC-aminotag phosphoramidites are reported here which have been designed, synthesized and evaluated. PC-aminotag phosphoramidite (Scheme 1, n = 0) consists of a protected amino group linked through a methylene group to a photocleavable 1-(2-nitrophenyl)ethyl linker derivatized with N,N[prime]-diisopropyl-2-cyano-ethyl phosphoramidite. PC-X-aminotag (Scheme 1, n = 1) contains an additional spacer (6-aminocaproic acid, referred to as `X') between the amino group and the photocleavable linker.Properties of modified oligonucleotides prepared using PC-aminotag phosphoramidites and their conjugates are studied and several applications of PC-aminotag phosphoramidites are described.
Figure 1.Absorption values measured at 490 nm for hybridization assay on CPG beads. Sample 1, BIO-PC-X-probe; sample 2, 1 BIO-PC-X-probe, irradiated after hybridization; sample 3, 1 BIO-PC-X-probe irradiated before hybridization; sample 4, no biotinylated probe added; sample 5, no enzyme added. Scheme 1. Structure of PC-aminotag phosphoramidates. All chemicals used in the synthesis were purchased from Aldrich Chemical Co. (Milwaukee, WI), unless otherwise indicated. Biotinamidohexanoic acid NHS ester was purchased from Sigma (St Louis, MO), digoxigenin-X-NHS ester (digoxigenin-3-O-methylcarbonyl-[epsis]-aminocaproic acid N-hydroxysuccinimide ester) was obtained from Boehringer-Mannheim (Indianapolis, IN) and tetramethylrhodamine-X-NHS ester [6-tetramethylrhodamine-5-(and-6)-carboxamidohexanoic acid succinimidyl ester, TAMRA-X-NHS] was from Molecular Probes (Eugene, OR). Fmoc-NHS and N-Fmoc-6-aminocaproic acid were from Novabiochem (La Jolla, CA). DNA synthesis, HPLC and photocleavage conditions were performed as described before (14). The synthesis of PC-aminotag-phosphoramidites was performed similar to the synthesis of PC-biotin-phosphoramidite (14). The detailed synthesis of these compounds will be published elsewhere (J.Olejnik, E.Krzymanska-Olejnik and K.J.Rothschild, manuscript in preparation). Syntheses of 5[prime]-PC-amino-olignucleotides were carried out as described before for PC-Biotin phosphoramidite (14). Syntheses were carried out in the Trityl-On mode. Coupling efficiency for PC-aminotag phosphoramidites was determined by HPLC analysis of several sequences and found to be in the range of 95-98%. The molecular mass of 5[prime]-PC-amino-(dT)7 and 5[prime]-PC-amino-X-(dT)7 was verified before and after photocleavage reaction by MALDI-TOF, (values in brackets are calculated): 5[prime]-PC-amino-(dT)7 2326.3 (2326.6), uncleaved; 2148.4 (2147.4), cleaved; 5[prime]-PC-amino-X-(dT)7 2438.4 (2439.7), uncleaved; 2147.2 (2147.4), cleaved. Control 5[prime]-phosphorylated sequences were synthesized using the chemical phosphorylation reagent PhosphalinkTM (Applied Biosystem, Foster City, CA) according to the manufacturer's instructions. Crude 5[prime]-PC-amino-d(T)7 or 5[prime]-PC-amino-X-d(T)7 (5.0 OD) was dissolved in 200 µl of 0.1 N NaHCO3 (pH = 8.5). To this solution was added 100 µl of the NHS ester (biotin, digoxigenin or rhodamine) solution in anhydrous DMF (10 mg/ml). The solution was incubated overnight at room temperature, then it was purified by gel filtration (Sephadex G-25, NAP-10 column, Pharmacia, Piscataway, NJ), freeze dried and purified to homogeneity on RP HPLC. For photocleavage studies, a solution of 0.2 OD of oligonucleotide in 50 µl of H2O was irradiated for 5 min in a clear polypropylene microcentrifuge tube and a 20 µl aliquot was then analyzed by HPLC. Hybridization probe (5[prime]-PC-amino-X-TCG-AGA-GGT-ACA-TCG-TG-3[prime]) was synthesized, labeled with biotin-NHS ester and purified by HPLC. The complementary sequence was prepared on a C27 support from Peninsula Laboratories (Belmont, CA). For hybridization, 10 pmol of probe was hybridized with ~4 nmol of target sequence on CPG beads. Hybridization conditions: 25 mM NaCl, 20 mM Tris-HCl, 10 mM MgCl2, pH = 7.5; denaturation 72°C, 10 min, annealing: 72-37°C over 30 min. The beads were washed with 100 µl wash buffer (2 M NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH = 8.0) containing 2% w/v BSA, supernatant removed and 200 µl of streptavidin-horseradish peroxidase (0.05 µg/ml) added. The mixture was incubated for 1 h, centrifuged briefly and supernatant removed. The beads were washed with 200 µl of wash buffer (15 min, 4 times). Finally, 250 µl of o-phenylenediamine (OPD) in stable peroxide buffer (Pierce, Rockford, IL) was added and the mixture incubated for 20 min. An aliquot of 250 µl of 2 M H2SO4 was added and the OD value at 490 nm was measured. Two oligonucleotides, 44mer [5[prime]-H2N-PC-GGC-CGC-TAC-CTC-TCA-(CCA)6-CTG-ATG-AGC-T-3[prime]] and 35mer [5[prime]-H2N-PC-CAT-CAG-(TGG)6-TGA-GAG-GTA-GC-3[prime]] were synthesized at 0.2 µmol scale with 5[prime]-PC-aminotag modifier. These were cleaved and deprotected using standard procedures and then freeze-dried. 6-aminohexanoic acid-NHS agarose (100 mg) (Sigma, St Louis, MO) was suspended in 300 µl of water, vortexed briefly and spin-filtered using a 0.22 µm Ultrafree MC filter (Millipore, Bedford, MA), then resuspended in 300 µl of water. Crude 5[prime]-PC-amino-oligonucleotide (10.0 OD) was added to a suspension of activated agarose, followed by 40 µl of 1 M NaHCO3. The mixture was vortexed at room temperature for 1 h, spin-filtered and washed with 100 µl of wash buffer (2 M NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH = 8.0). Finally, the beads were resuspended in 300 µl of wash buffer and irradiated with near-UV light for 5 min. After irradiation, the suspension was spin-filtered and the beads were washed with wash buffer (100 µl). The combined filtrates were then desalted on a NAP-10 column, analyzed by UV absorption spectroscopy and by denaturing PAGE. PC-aminotag phosphoramidites (Scheme 1) were designed for direct use in any automated DNA/RNA synthesizer employing standard phosphoramidite chemistry. The selective reaction of PC-aminotag phosphoramidites with the free 5[prime]-OH group of a growing oligonucleotide chain, followed by cleavage from the support and deprotection, results in the introduction of a phosphodiester group linked to a primary aliphatic amino group through a photocleavable linker. This amino group can then be used to introduce a variety of photocleavable markers through a postsynthetic modification reaction (Scheme 2) with amine reactive reagents. It can also be used to attach synthetic oligonucleotides to activated solid supports for a variety of applications requiring immobilization and release of DNA molecules. Upon irradiation with near-UV light, the phosphodiester bond between the photocleavable linker and the phosphate is cleaved, resulting in the formation of a 5[prime]-monophosphate on the released oligonucleotide. The 1-(2-nitrophenyl)ethyl moiety is converted into a 2-nitrosoacetophenone derivative and is released together with the attached marker molecule M. Scheme 2. Reaction of PC-aminotag modified oligonucleotide and subsequent photocleavage. Figure 2. 20% polyacrylamide electrophoresis gel of 44mer (lanes 1-3) and 35mer (lanes 4-6) visualized by UV-shadowing, each lane representing 0.25 OD260 units of material. Lanes 1 and 4, crude 5[prime]-aminotag-oligonucleotides; lanes 2 and 5, purified and photoreleased oligonucleotides; lanes 3 and 6, filtrate containing sequences not reacted with activated agarose. The heptamers 5[prime]-PC-amino-(dT)7 and 5[prime]-PC-amino-X-(dT)7 were assembled using PC-aminotag-phosphoramidites in an automated DNA/RNA synthesizer. The unmodified sequence, 5[prime]-OH-(dT)7, and a 5[prime]-phosphorylated sequence, 5[prime]-p-(dT)7, were prepared using standard procedures (Materials and Methods). Table 1 shows retention times for unmodified 5[prime]-PC-amino-(dT)7, 5[prime]-PC-amino-X-(dT)7 as well as for their conjugates prepared with biotin (BIO), digoxigenin (DIG) and tetramethylrhodamine (TAMRA) NHS esters. In both 5[prime]-PC-amino-(dT)7 and 5[prime]-PC-amino-X-(dT)7, two peaks are observed, which can be attributed to the introduction of chiral 1-(2-nitrophenyl)-ethyl moiety onto the oligonucleotides. These peaks appear at 17.5 and 18.3 min for 5[prime]-PC-amino-(dT)7 and at 19.2 and 19.9 min for 5[prime]-PC-amino-X-(dT)7 compared to unmodified 5[prime]-OH-(dT)7 which appears at 14.2 min. This shift can be attributed to the introduction of hydrophobic photocleavable moiety on 5[prime]-PC-amino-(dT)7 and the presence of additional hydrophobic spacer arm in case of 5[prime]-PC-amino-X-(dT)7 which further increases the retention time. The reaction of 5[prime]-PC-amino-(dT)7 with biotin-X-NHS results in formation of a product with the retention times increased to 24.3 and 24.9 min compared to unmodified 5[prime]-PC-amino-(dT)7. This is in good agreement with a retention time observed previously for the same conjugate prepared using PC-biotin phosphoramidite (14). The reaction of digoxigenin-X-NHS ester with 5[prime]-PC-amino-(dT)7 results in a product with one peak at 32.4 min. This is most likely due to high hydrophobicity of the digoxigenin moiety. The modification of 5[prime]-PC-amino-(dT)7 with tetramethylrhodamine-X-NHS ester results in three peaks with retention times of 29.3, 29.8 and 31.9-32.2 min. This complex pattern is probably due to the presence of positional isomers (C-5 and C-6) in tetramethylrhodamine NHS ester. The reaction of 5[prime]-PC-amino-X-(dT)7 with biotin-X-NHS, digoxigenin-X-NHS and tetramethyl-rhodamine-X-NHS ester gives very similar results, but with slightly longer retention times due to the presence of additional hydrophobic spacer arm X in the conjugate. Table 1. Retention times of PC-amino-d(T)7, PC-amino-X-d(T)7 and their conjugates with biotin (BIO), digoxigenin (DIG), tetramethylrhodamine (TAMRA) before and after irradiation The effects of irradiation of 5[prime]-PC-amino-(dT)7 (5[prime]-amino-PC-dT7) and 5[prime]-PC-amino-X-(dT)7 (5[prime]-amino-PC-X-dT7), as well as their conjugates, are also presented in Table 1. The solution of 5[prime]-PC-amino-(dT)7 and 5[prime]-PC-amino-X-(dT)7 were subjected to near-UV irradiation for the period of 5 min and then analyzed by RP-HPLC. In all cases after irradiation, only one peak can be observed at ~13.3-13.5 min. This retention time is in good agreement with the retention time of the phosphorylated control, 5[prime]-p-(dT)7 (13.4 min). This result indicates that upon exposure to near-UV light, the photocleavable label introduced onto 5[prime]-end is quantitatively photocleaved, resulting in 5[prime]-p-(dT)7. In order to evaluate the modification reaction as well as the photocleavage reaction in a highly-sensitive hybridization assay, a PC-aminotag modified probe was synthesized and labeled with biotin-NHS ester. In this case, a 17mer PC-amino-oligonucleotide was utilized which contained all four bases (Materials and Methods). The introduction of bases containing exocyclic amino groups is important in order to evaluate their potential reactivity toward NHS esters such as biotin-NHS. In the case of such a reaction, the probe would not be photoremovable and therefore interfere with subsequent probe assays. After HPLC purification, the PC-biotin-oligonucleotide probe was hybridized with a complementary sequence immobilized on CPG beads followed by colorimetric (streptavidin-HRP/OPD) detection. The results of this experiment are presented in Figure In order to evaluate the reactivity of PC-amino-oligonucleotides with activated supports, and to evaluate the usefulness of PC-aminotag-phosphoramidite for photocleavage-mediated affinity purification/phosphorylation of oligonucleotides, two 5[prime]-PC-aminotag labeled sequences, a 44mer and a 35mer, were prepared. After deprotection, the crude 5[prime]-PC-aminotag oligo-nucleotides were separately incubated with NHS-activated agarose beads. The beads were then washed, resuspended and finally irradiated to obtain the full-length phosphorylated oligonucleotides. Figure In a previous paper, we described photocleavable biotin phosphor-amidite which adds a photocleavable biotin to the 5[prime]-end of synthetic oligonucleotides (14). Through the use of streptavidin affinity media, PC-biotin-phosphoramidite provides a rapid method for the purification, isolation and phosphorylation of synthetic oligonucleotides. In the present work we have extended the scope of this approach by introducing PC-aminotag phosphor-amidites. These reagents introduce a primary amino group attached to a photocleavable linker onto the 5[prime]-end of synthetic oligonucleotides. Similar to conventional amino modifiers phosphoramidites (2-4), these photocleavable amino groups can be modified with numerous amine reactive reagents including fluorescent tags (rhodamine), haptens (digoxigenin) and ligands for binding proteins (biotin). However, unlike `aminolinkers', PC-aminotags also provide a means for rapid removal of the label resulting in an unmodified 5[prime]-phosphorylated oligonucleotide. In general, PC-aminotag phosphoramidites provide expanded applications for the use of markers and tags introduced into DNA/RNA. A variety of applications have already been described for aminoalkyl-terminated synthetic oligonucleotides. These include the introduction of a ligand or probe for the purpose of non-radioactive detection of specific DNA/RNA sequences, conjugation to solid supports for the purpose of affinity capture, and conjugation to peptides and proteins for intracellular delivery. However, in none of these cases can the probe be easily removed or the covalent link broken without the use of chemical reagents which having many disadvantages. In contrast, PC-amino-linked probes or oligonucleotide attachments to solid supports can be rapidly cleaved by the application of near-UV light. There are a variety of DNA/RNA related applications where photocleavage of an aminolinker either to a substrate or marker molecule is attractive. For example, DNA/RNA analysis with hybridization probes often involves the detection of several unique sequences within the target sample. However, such multiple detection schemes require the removal of the `read-out' portion of the detection complex prior to sequential reprobing with a second hybridization probe. Usually, the labeled probe is stripped from the membrane using high temperature and denaturing agents (15). A probe conjugated to the label via PC-aminotag linker should facilitate much easier removal of the label/enzyme complex by simply illuminating the sample with an appropriate UV source. In principle, a large number of DNA/RNA sequences could be detected sequentially in the same sample. In a second example, a photoreleasable molecule with a defined molecular weight (e.g. mass markers) conjugated to hybridization probes via PC-aminotag can facilitate multiplex detection of several sequences in the target DNA/RNA by mass spectrometry (J.Olejnik, E.Krzymanska-Olejnik, H.-C.Lüdemann, F.Hillenkamp and K.J.Rothschild, manuscript in preparation). This has many advantages over direct detection of oligonucleotide hybridization probes (16), since the sensitivity of MALDI-MS to detect oligonucleotides decreases rapidly as a function of mass (17,18). This occurs due to the formation of adducts of alkali ions with the sugar-phosphate backbone and the fragmentation of DNA during MALDI. In contrast, mass markers consisting of a series of peptides are not prone to these problems and can thus be used to identify the presence of a specific sequence in the immobilized target DNA/RNA. A variety of promising applications including the development PC-aminotag-mass markers are currently being investigated for this purpose (19). The work was supported by SBIR grants to AmberGen, Inc. from the National Institutes of Health (2R44 GM54920-02) and the Army Research Office (ARO-DAAH04-96-C-0050) and by a University Research Initiative grant from the Army Research Office (ARO) (DAAL03-92-G-0172) to K.J.R. The authors wish to thank Stephanie Hahner and Hans-Christian Lüdemann (University of Münster, Germany) for help with MALDI-MS spectra and Andrew Muir for proofreading.
This article has been cited by other articles:
MATERIALS AND METHODS
Synthesis of photocleavable aminotag phosphoramidites
5[prime]-PC-amino-oligonucleotide synthesis
Reaction with activated marker molecules and photocleavage
Hybridization and colorimetric detection
Reaction of 5[prime]-PC-amino-oligonucleotide with activated support and photocleavage
RESULTS
Design of photocleavable aminotag-phosphoramidites
Synthesis and evaluation of PC-amino-oligonucleotides and their conjugates
Evaluation of a PC-amino-oligonucleotide in hybridization assay
Reaction of PC-amino-oligonucleotides with activated supports and photocleavage-mediated affinity purification
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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