Bioconjugate Chem. 1903, 4, 386-394
306
Photoaffinity Behavior of a Conjugate of Oligonucleoside Methylphosphonate, Rhodamine, and Psoralen in the Presence of Complementary Oligonucleotides John Thaden and Paul S. Miller' Department of Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. Received March 30, 1993"
A 3'-[[2-[N-(3-aminopropyl)-N-(2-hydroxyethyl)amino1ethylphosphoryl1oligodeoxyribonucleoside methylphosphonate 12-mer was synthesized using the Aha-CPG solid support [Thaden, J. and Miller, P. S. (1993) Bioconjugate Chem., companion paper in this issue]. The oligomer was conjugated at the 3' primary aliphatic amine with tetramethylrhodamine 5-isothiocyanate. The rhodamine linker/spacer was stable in 10% fetal calf serum. After enzymatic phosphorylation, the molecule was conjugated at the 5' phosphate with 4'-[N-(2-aminoethyl)aminomethyll-4,5',8-trimethylpsoralen [(ae(AMT)], The rhodamine/psoralen doubly-conjugated oligomer formed photoadducts with complementary singlestranded DNA and RNA oligonucleotides when irradiated with long-wavelength ultraviolet light. The efficiency of UV cross-linking slightly exceeded that of a colinear, psoralen-derivatized oligonucleoside methylphosphonate, and exhibited relationships with UV fluence and temperature that are characteristic for psoralen-conjugated methylphosphonates. The 1:lcomplex formed with the oligodeoxyribonucleotide target could be detected by its red fluorescence. Mouse L949 cells grown in the presence of the double conjugate were shown by means of computer-assisted epifluorescence microscopy to have internalized it. There was an accumulation of intensely fluorescent points and spots in a juxtanuclear region of the cytoplasm, and a faint, diffuse signal in the entire cell area.
Previous reports from our laboratory and elsewhere have demonstrated that psoralen-conjugated oligonucleoside methylphosphonates can effectively cross-link to singlestranded DNA targets and to mRNA in vitro upon activation with long-wavelength UV light (Lee et al., 1988a,b; Kean et al., 1988; Bhan and Miller 1990; Isaacs et al., 1989; Levis, 1993). Psoralen-conjugated oligomers were also shown to be effective,sequence-specificinhibitors of herpes simplex virus replication in infected cells (Kulka et al., 1989) and of p21 synthesis in ras oncogene transformed NIH 3T3 cells (Chang et al., 1991). For mechanistic studies in cell culture, the utility of psoralen-derivatized oligonucleosidemethylphosphonates could be considerably enhanced if they were conjugated to an easily detectable fluorescent label. The tetramethylrhodamine fluorophore is strongly excited by the 546 nm emission of a mercury arc lamp, resists photobleaching, is not readily obscured by cellular autofluorescence (Simon and Taylor, 1986;Benson et al., 19791,is insensitive to pH in the physiological range (Geisow, 1984), and has little spectral overlap with the photoactivation and emission spectra of psoralen compounds. In this paper we describe the synthesis of a psoralenl rhodamine doubly-conjugated oligonucleoside methylphosphonate. We demonstrate that the linkage to rhodamine is stable under cell culture conditions, that the oligomer efficiently cross-linkswith single-stranded RNA and DNA targets when irradiated with near UV light, that the rhodamine fluorescencesurvives UV irradiation and crosslinking to a target, and that the oligomer is readily taken up by cells in culture.
* Author to whom correspondence should be addressed. e Abstract
published in Advance ACS Abstracts, September
1, 1993. 1043-1802/93/2904-0386$04.00/0
EXPERIMENTAL PROCEDURES
Reversed-phase HPLC was performed as described (Bhan and Miller, 19901, on either Rainin Microsorb C18 80-215-C5 or Whatman ODS-3 RAC I1 columns for analytical purposes and on Whatman ODS-3RAC columns for preparative work. These were eluted at rates of 1,1.5, and 4 mL min-l, respectively, using linear acetonitrile gradients of slope 1.5% min-l for analytical runs and 0.75 7% min-l for preparative runs. For HPLC with multiplewavelength detection, a RAC I1 column was mounted on a Waters 600E system with a Model 990 photodiode-array detector. Two denaturing PAGE procedures were used: (1)15 or 20 % polyacrylamide gels (acrylamide/bisacrylamide 19:1,0.75 X 170 mm) containing 7 M urea and TBE (Maniatis et al., 1982)were electrophoresed at 35 V cm-l; (11) gels were formulated as described except TBE was replaced by TPE buffer (0.089 M Tris base, 0.089 N phosphoric acid,0.1 mM EDTA) and 33 mM dithiothreitol. The presence of dithiothreitol greatly retards fluorescence fading (Picciolo and Kaplan, 1984). TPE-buffered gels were electrophoresed at 20 V cm-l. Rhodamine fluorescence in gels was detected either under room light or, for faint bands, with the gel on a short-wavelength UV transilluminator. The latter was used during fluorophotography with Polaroid 665 film and a deep yellow filter (e.g. Wratten #9, Eastman, Inc.), as for photography of ethidium bromide gels. Autoradiography was done with XAR-5 film (Eastman) at -20 OC. Solvent removal was by evaporation in uucuo (oil pump) at not more than 40 OC. Desalting of oligomers was accomplished with SepPak Cla cartridges (Waters) prewashed with acetonitrile (10 mL) followed by 50% acetonitrile (5 mL) and, unless noted, 5% acetonitrile in 0.1 M sodium phosphate (pH 5.8,10 mL). Samples were loaded as solutions in more of the final prewash buffer. Loaded cartridges were washed with water (20 mL). Desalted products were eluted with 50% acetonitrile (4 mL), and solvents were removed. All @ 1993 American Chemical Society
BloconJugfite Chem., Vol. 4, No. 5, 1993 387
Rhodamine/Psoralen Conjugated Oligomers
Scheme 1. Synthesis of a Rhodamine/Psoralen DoublyConjugated Oligonucleoside Methylphosphonate. R
o=
R’
8
Protected Aha-CPG
-0-
. ,
b-
nucleoside synthons o (a) Automated synthesis on support 6; (b) hydrazine hydrate/ HOAc/pyr; (c) ethylenediamine/95% EtOH; (d) 80%HOAc; (e) A T P / F polynucleotide kinase/kinase buffer/MeCN; ( f )TRITC isomer G/bicineNaOH buffer, pH 9/EtOH, (9)EDAC/imidazole HCl buffer, pH 6/MeCN (h) (ae)AMT/2,6-lutidine-HCl buffer, pH 7.5/MeCN.
operations with substances containing rhodamine and/or psoralen were conducted under red or subdued light. Rhodamine-modifiedoligomers were stored dry at -20 OC. Operations involving RNA were done using diethylpyrocarbonate-treated liquids (Maniatis et al., 1982)and ovenbaked labware. Synthesis of Substances Illustrated in Scheme I. 3’-(Aha1-phosphoryl)oligodeoxyribonucleoside Methylphosphonate 1 (Scheme I). Substance 1 was prepared upon the Aha-CPG support as described in a companion paper in this issue (Thaden and Miller, 1993) using nucleoside 2-cyanoethyl phosphoramidite synthons for the first and last couplingsand methylphosphonamidite synthons for all others. 5’-Phosphoryl-3’-(Aha-phosphoryl)oligodeoxyribonucleoside methylphosphonate 2 (Scheme I). This kinase reaction was done first because the presence of a 5’-phosphate facilitated later HPLC purification. To a solution of oligomer 1 (1 pmol) in 50% acetonitrile (0.6 mL) were added 0.5 M Tris-HC1 buffer 0.05 M MgC12 (pH 7.6, 0.3 mL), 0.1 M ATP (0.1 mL), 50 mM dithiothreitol (0.3 mL), and water (1.7 mL) and, after swirling, Tqpolynucleotide kinase (10 pL, 300 units). The reaction solution was incubated at 37 O C , with swirling gently every 15 min. More kinase (5 pL) was added at 30 and 60 min, the last bolus preceded by dropwise addition of acetonitrile (180 pL). At 90 min, the reaction solution was diluted with 0.2 M sodium phosphate (pH 5.8,l vol) and desalted. Phosphorylation went to completion as assayed by HPLC. Abbreviations used (ae)AMT, 4’-[[N-(a-aminoethyl)amino]methyl]-4,5’,&trimethylpsoralen;Aha, N-(3-aminoprop-l-yl)N-(2-hydroxyethyl)-2-aminoethyl; CCD, charge-coupled device; DIC, differential interference contrast (Nomarski) optics; EDAC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; EDTA, ethylenediaminetetraacetic acid, 0.8 M stock, pH 8.0; EMEM, minimal essential medium with Earle’s salts; FBS, fetal bovine serum, not heat-treated; HBSS, Hank’s buffered saline solution; phosphate buffer, 0.1 M sodium phosphate buffer, pH 5.8; T,, temperature at which nucleic acid cross-linking by psoralenconjugated oligomers is half-maximal; TPE buffer, 0.089 M Tris base, 0.089 N phosphoric acid, 0.1 mM EDTA; TRITC isomer G, N,N’-tetramethylrhodamine 54sothiocyanate.
5’-Phosphoryl-3’- [N-[ 3-[[(NJV,N,N-tetramethylrhodaminyl-5-yl)thiocarbamoyl]amino]propl-yl)-N(2-hydroxyethyl)-2-aminoethyl]phosphoryl]oligodeoxyribonucleosideMethylphosphonate3 (SchemeI). TRITC isomer G (Molecular Probes, Inc., 5 mg, 10.4 pmol) was vortexed and sonicated with reagent alcohol (0.6 mL, Fisher Scientific) until dissolved. The dye solution was immediately added to a solution of oligomer 2 (1pmol) in bicineaodium hydroxide buffer (0.2 M, pH 9.0, 1 mL) and reagent alcohol (0.4 mL). The reaction was stirred in the dark for 18 h. Solvents were evaporated, and the residue was dissolved in a minimal volume of 0.1 M sodium phosphate buffer (pH 5.8) containing 25 % acetonitrile. The solution was loaded onto a Sephadex G-15column (1.6X 20 cm). Products were eluted with the same buffer. The first red peak was collected and freed of acetonitrile under vacuum. Product 3 was separated from unreacted oligomer 2 by preparative HPLC and both fractions were desalted. Conjugation yield was typically 85% as determined by HPLC peak area. 5’-(ae)AMT-3’-[ [N-[3- [[(N,N,N’,N’-tetramethylrhodamin-5-yl)thiocarbamoyl]amino]prop-1-yl]-N(2-hydroxyethyl)-2-aminoethyl]phosphoryl]oligodeoxyribonucleosideMethylphosphonate4 (SchemeI). Following the adaptation of Bhan and Miller (1991) of the two-step procedure of Chu et al. (19831, substance 3 (40 A254 units, 0.3 pmol) was dissolved with acetonitrile (120 pL) and 0.1 M imidazole-hydrochloride buffer (pH 6.0, 0.6 mL). Freshly made 1 M EDAC (80 pL) was added. The reaction was allowed to proceed at 22 OC in the dark for 3 h. A Sep-Pak cartridge was prepared, but using 0.2 M bicine-sodium hydroxide (pH 9.0) containing 10% ( u l u ) acetonitrile for the final prewash solvent. The reaction solution was diluted with 0.8 mL of the same solvent, loaded onto the column, and desalted. The dried, crude imidazolide was dissolved with acetonitrile (66 p L ) and 0.25 M 2,6-lutidine hydrochloride (pH 7.6, 280 pL) and reacted with 0.05 M (ae)AMT (70 pL) in the dark for 3 d at 22 OC. After desalting by the normal protocol, products were separated by denaturing PAGE and visualized under subdued room light. To identify compound 4 among several red bands, all but extremely minor bands were excised from the gel. Gel pieces were macerated. The pastes were steeped in 25 5% acetonitrile (3 mL, 2 h). After low-speed centrifugation, supernatants were sampled for the following test: Samples were treated with 0.1 N hydrochloric acid (1 h, 37 “C). The acid was removed by coevaporation with water. Products were resolved by denaturing PAGE alongside a lane of authentic compound 3 and visualized by fluorophotography. Acid converted only the most abundant psoralen conjugation product (Rf = 0.65 relative to compound 3) to a species that comigrated with 3 on gels. The macerated gel piece containing the Rf 0.65 species was eluted twice more as above. The pooled eluate was diluted with 0.35 M sodium phosphate buffer (pH 5.8,2.5 vol). The entire volume was loaded onto 8 Sep-Pak cartridge for desalting. Other Synthetic Oligomers. The 5’-(ae)AMT-oligonucleoside methylphosphonate 5, (Scheme I), oligodeoxyribonucleotide 6 (Chart I), and oligoribonucleQtide7 (Chart I) were generously donated by Dr. J. M. Kean. The nucleic acid targets were enzymatically phosphorylated using [y-32PlATPand polynucleotide kinase as described (Miller et al., 1991). 5’-[32Pl-labeled compound 3 was prepared by sequential reactions with calf intestinal phosphatase and polynucleotide kinase (Chaconas and van de Sande, 1980, method 3).
388
Thaden and Mlller
Bioconjtgate Chem., Vol. 4, No. 5, 1993
In Vitro Cross-Linking. Two procedures were used, both adapted from the assay of Lee et al. (1988b). Probe in Excess. The reactions were done in borosilicate glass tubes (10 x 75 mm, Corning). Each tube contained cross-linking buffer (50 mM Tris, 0.1 M sodium chloride pH 7.6,lO pL) in which had been dissolved either probe 4 or probe 5 (5 pM, 50 pmol) and radiolabeled oligonucleotide target 6 or 7 (0.1 pM, 1 pmol). The conjugate probe 5, not pictured, is identical with probe 4 in both base sequence and 5’-psoralen/linker structure, but it lacks the 3’-rhodamine/linker modification of 4. The stoppered tubes were equilibrated to a chosen temperature, then some of them were irradiated with a Blak-Ray Longwave UV lamp (UVP, San Gabriel) for 15 min as follows: The tubes were mounted on a rotator such that the center of the bulb face was parallel to the tubes’ sides, and at a distance varying with rotation from 6 to 10 cm measured to the bottom, center, of each tube. Rotation carried tubes through a water bath set at the pre-equilibration temperature, and also through the most intense spot of the lamp. Wavelengths shorter than 300 nm are absorbed by the borosilicate glass tubes. This geometry has been reported to deliver long-wave UV light to the solutions at a fluence rate of 8.3 kJ m-2 min-’ (Lee et al., 198813). The samples were then analyzed by PAGE procedure I and autoradiography of the wet gels. Isotopic bands were quantified by excision from the gel and scintillation counting. Equivalent regions from dummy lanes were likewise measured to determine background levels. Crosslinking was calculated as the percentage of the total counts above background that resided in gel-shifted bands. Target in Excess. Tubes contained cross-linkingbuffer (20 pL) in which was dissolved probe 4 (120 pmol or 160 pmol as specified) and radiolabeled DNA target 6 at twice the concentration of the probe. A 10-min pre-equilibration was followed by UV irradiation for 10minor for the interval specified. Samples were resolved by PAGE. After autoradiography and fluorophotography of the gels, isotopic bands were quantified. Percentage of cross-linking was calculated. Fluorophotograph negatives were laserscanned (LKB Ultroscan XL, x width = 4) to produced density plots of each lane. Plots were copied to 20 lb xerographic paper. Baselines were fitted using plots of empty lanes, and peaks were cut out with scissors and weighed. Cell Culture and Treatmentwith Oligomer. Mouse L929 fibroblasts (L cells) were maintained in 25 cm2culture flasks with 1:lO splits done twice weekly. The close distance between objective and condenser lenses on the microscope used to view living cells dictated the design of chambered slides upon which cells were grown and viewed. A hole (1.9 cm diam) was drilled in each of several stainless steel plates (7.6 cm X 5.1 cm X 1 mm). Cover glasses (#1’/2,22 mm square) were soaked in 6 N nitric acid, rinsed with deionized water, soaked in 0.1 mM EDTA, and again rinsed with deionized water. A cover glass was mounted over one side of the hole in each plate using silicone stopcock grease. Cylinders (1.2 cm high) were cut from a clear acrylic pipe (1.9 cm i.d., 2.6 cm 0.d.) and the cut surfaces were sanded smooth. A cylinder was affixed to the upper side of each hole using stopcock grease. The resulting chambers were placed in Petri dishes and autoclaved. Subconfluent L cells were trypsinized and seeded at one-fourth density into chamber assemblies each containing EMEM (0.5 mL) supplemented with 10% FBS. When the cellswere again subconfluent (48h), the medium was changed to EMEM containing 1%FBS and 10 pM of 4, prepared as follows: Compound 4 was sterilized in
70% ethanol, dried in uacuo, and dissolved in filtered EMEM lacking FBS. The solution was sonicated for 2 min, supplemented with FBS (to 1% 1, and used immediately. Control medium was prepared likewise, but omitting oligomer. The cells were incubated for 9 h. Fluoromicroscopy. The acryliccylinder was detached. Cells were rinsed several times with HBSS. A cover glass was pressed into the grease which had secured the cylinder, forming a 1 mm thick chamber filled with HBSS (-0.3 mL). Cells exhibited no morphological changes for at least 40 min in these chambers during microscopy a t ambient temperature without buffer refreshment. The chambered slide was placed on the stage of an inverted microscope outfitted with both epifluorescence and differential interference contrast (DIC) optics, a CCD camera (Callahan et al., 1992), and a dedicated image processing system (Perceptics Corp., Knoxville, TN, Model 9210-VI).2 Cells viewed through a lOOX oil immersion objectivewere located and brought into focus using transmitted light and DIC optics. To limit bleaching, a gray filter (0.03, 97% attenuation) and a deep yellow filter (Wratten #9, Eastman) were inserted in the light path, illumination was limited by the field diaphragm to the central two-thirds of the viewed area, and exposures were kept brief. The microscope was switched to epifluorescence optics using a filter set designed for rhodamine fluorescence. Without altering the focus, the camera was activated and the portion of the view framed by the camera was excited for 5 s by incident illumination from a 100-W mercury arc light, which was 90% attenuated by a gray filter (0.1). The microscope was switched back to DIC optics and a second digital image was captured during 2 s of transillumination by white tungsten filament light, 97 5% filter-attenuated. The stage was moved to a new field of cells located at least five field-diameters from the prior one, and the process was repeated. Ten to 40 different optical fields were imaged in this manner from cell lawns originally grown in each chamber. Image Processing. Algorithms for processing images of cells were implemented on a Macintosh computer using HyperScope (Perceptics, Inc.), an extension to the Hypercard programming language. Each image was an array of 512 X 384 pixels. Pixels collected photons from contiguous square areas (0.23 pm per side with the lOOX lens) on the surface of the chambered slide. After mathematical application of a shading correction (Aikens et al., 1989) to images, the CCD camera pixels were seen be linear throughout a dynamic range of over 2.5 log units of fluorescence intensity (Callahan et al., 1992). The following shading correction formula was used3 in
I- (CD/lO) 10
- (CDIlO)
where I is the raw image, I‘is the corrected image, L is a luminous field mean image formed by averaging ten images of various optical fields on a chambered slide loaded with a solution of rhodamine B (A557 = 0.005)and 33 mM Additional details of the microscope, optics, light sources, CCD camera, image processor, and processor-related software are described in a manuscript in preparation [Callahan, D. E., Ts’o,P. 0. P., and Lesko, S. A. (1993) Exp. Cell Res.]. Image shading correction was necessitated by a “hot spot” of epi-illumination caused by imperfect diffusion of the arc image, and “vignetting” introduced by the microscope optics. It also removes interpixel differences in camera performance.
Bloconlugte Chem., Vol. 4, No. 5, 1893 S89
Rhodamine/Psoralen Conjugated Oligomers
Chart I. Base Sequence of Methylphosphonate 1-5 and Phosphodiester 6,7 Oligomers" D-5'AAT T G A C A A A T C C T A T T T T3'6 3'CI G I I I A G G A I A 5 ' - D 1-5 R-"AAU U G A C A A A U C C U A U U UU3'7
Underlined letters represent nucleosides with 3'-methylphosphonate linkages. a
dithiothreitol, and D is each of ten dark current images, captured with the camera shielded from light.4 These calibration images were captured during the same session and at the same shutter speed as the experimental images of cells. Each DIC image was scaled so its brightest pixel was white, sharpened by spatial masking methods (Gonzalez and Wintz, 1987), and registered in the x-y plane to the matching fluorescence image by the alignment of features, typically cell processes and edges of nuclei, which were visible in both images. Registration usually involved horizontal or vertical motion of the DIC image by less than 3 pixels. For color photographs, fluorescence images were displayed in red while the corresponding DIC images were overlaid at one-third intensity in both green and blue. The computer monitor screen was photographed. RESULTS AND DISCUSSION
Synthesis of the Fluorescent Oligomer. Using a special controlled pore glass solid support (Aha-CPG) described elsewhere in this issue (Thaden and Miller, 19931, we synthesized an oligonucleoside methylphosphonate which had a base sequence (Chart I) complementary to nucleotides 165-176 of vesicular stomatitis virus matrix protein mRNA (Rose and Gallione, 1981) and a 2-(tertaminoethy1)phosphoryl structure at its 3' end. The 3' modification provided a primary aliphatic amine for subsequent conjugation with an amine-reactive fluorophore. We enzymatically phosphorylated the oligomer's 5' end and then reacted the oligomer with excess tetramethylrhodamine 5-isothiocyanate. It was necessary to alter the buffer used in this reaction because standard isothiocyanate reaction buffers (sodium bicarbonate, borate, or phosphate at pH 8.5-9.5) produced ladders of oligomer degradation products on PAGE gels. In bicine buffer (pH 9), the result was an intact oligomer,joined a t its 3' end to tetramethylrhodamine by a linkerlspacer containing thiocarbamoyl and phosphodiester linkage^.^ Stability of the 3' End Modifications. To see if the conjugate was stable under cell culture conditions, a solution of compound 3 in growth medium containing 10?4 fetal bovine serum was incubated at 37 OC and sampled at 3.5 and 24 h. HPLC with multiple wavelength detection was performed on these samples, on untreated conjugate 3, and on the medium alone (Figure 1). The chromatographic profile for untreated conjugate 3 (Figure la) revealed a species which absorbed at both 254 and 540 nm Unlike bright-field images, dark-current images were dim, i.e. used few of the 65 536 gray levels. Hence, they were low in precision and high in noise. These problems were minimized by the summation of ten dark-field images. The gain in precision was preserved by relocating the averaging step (division by ten) to the flat-fielding formula, which could be implemented using floating point arithmetic. In attempta to conjugate N-hydroxysuccinimides and other active esters of a xanthine dye to an oligomer with the same 3' modification as compound 3, we were thwarted by the appearance of a nonfluorescent major product.
2
L.
0
4
a Tr
-.--._.
.....,...___Y..._.__.._._..(._.._._....,
10
Minutes
20
~
"
...... ...
30
Figure 1. Reversed-phase HPLC study with detection at both 254 (-) and 540 nm (. of (a) 5'-phosphorylated, 3'-rhodamineconjugated oligonucleoside methylphosphonate (3); (b) EMEM cell culture medium containing 10% fetal bovine serum, and (c) 3.5-h and (d) 24-h incubations of a solution of compound 3 in like medium, at 37 "C. Arrows point to eluted species which change in amount during incubation in the presence of serum. HPLC: 45 mL linear 1-46% acetonitrile gradient in 0.1 M sodium phosphate, pH 5.8, 1.5 mL m i d . e)
and which eluted 20 min after the start of the gradient (upward arrow). A chromatogram of the medium alone (Figure lb) revealed several A264 components of the serum eluting during the first 10 min of the gradient, and the phenol red indicator dye eluting at 18.5 min, which absorbed weakly at 540 nm also. When compound 3 was incubated with serum-containingmedium for 3.5 h (Figure IC),the single conjugate 3 peak eluting at 20 min developed a shoulder on its trailing edge (downward arrows). The peak and its shoulder were evident at both 254 and 540 nm, and the area ratio between them appeared the same at both wavelengths. When incubation had proceeded for 24 h (Figure Id), the conjugate 3 peak eluting at 20 min appeared to have been replaced by a peak of equal size eluting 40 s later, at the position where the shoulder had been. No other peaks were found at either incubation time which were not also found in medium alone. The 40-s delay in HPLC elution time was identical with that produced during removal of the 5'-phosphate from conjugate 3 by reaction with calf intestinal phosphatase (not shown). In separate experiments, we and others have shown that 32P-labeledoligonucleoside methylphosphonates are dephosphorylated in serum-containing medium (unpub1ished;Akhtaret al., 1991),witharatethatisslower than for phosphodiester oligomers and variable, depending on serum lot and media storage conditions.6 We did not see evidence of degradation other than 5'-dephosphorylation: One would expect exo- or endonucleolytic cleavage of the methylphosphonate backbone to give a melange of earlier-eluting species a t the expense of the major peak. This result was not seen nor was it expected, since nuclease resistance of the methylphosphonate linkage has been established (Agrawal and Goodchild, 1987; Akhtar et al., 1991). One would expect a scission a t any bond in the linkerlspacer to uncouple the elution of the major A254 and A540 components of conjugate 3 during HPLC. This also was not seen. If, by chance, a linker/spacer scission were to generate decay product with chromatographic elutions identical to the starting compound, then such a scission might elude detection. Since 5'-dephosphorylation shifted the coincident peaks of 254 and 540 nm absorbance without unmasking a chance coelution of decay products, a more reasonable conclusion Medium for this experiment had been sterility-tested overnight at 37 O C and then stored at 4 "C for 2 weeks.
390 Bioconjugte Chem., Vol. 4, No. 5, 1993
is that the linkage between the oligomer and the rhodamine dye was resistant to nucleases and other degradative enzymes present in the medium containing 10% fetal bovine serum. Attachment of the Psoralen. In two steps, the 5'phosphate of conjugate 3 was reacted with the aminomodified psoralen (ae)AMT, in order to furnish the oligomer with a photoreactivecross-linking capability.This modification also protects the 5' terminus from nucleasecatalyzed hydrolysis. To identify substance 4 among several fluorescentproducts resolved by denaturingPAGE, advantage was taken of the fact that phosphoramidate linkages like that in compound 4 are cleaved by acid to the corresponding phosphate, which in this case would be the starting compound 3, whereas side products with unusual linkages, modified bases, altered 3' structures, etc., would not be expected to revert to starting material. Only the most abundant product reverted to a species with electrophoretic behavior like that of substance 3 (not shown). On this evidence, it was isolated and used in further experiments as conjugate 4. A colleague has shown that this linkage of (ae)AMT to oligomers is stable in medium containing 10% serum during incubation a t 37 "C for 24 h (Levis, 1993). When administered to cells in culture for various times, 32Plabeled, 5'-psoralen-~onjugatedmethylphosphonates can be recovered largely intact from cell lysates a t 4 h (unpublished). The majority is intact in both lysates and the overlying medium a t 12 h; however, there is significant degradation a t 24 h (Levis, 1993). Effect of the 3' End Modification upon CrossLinking (Probein Excess). Buffered salt solutions were prepared which contained either the 3'-rhodamine/5'psoralen doubly conjugated methylphosphonate 4 or the colinear 5'-psoralen conjugated methylphosphonate 5 in excess over either end-labeled DNA (6) or an analogous RNA single-stranded 19-mer target (7). Each solution was apportioned to four tubes. After equilibration in ice water for 1h, two of the four tubes in each set were UV irradiated a t 4 "C. The sixteen samples were analyzed by PAGE, followed by autoradiography (Figure 2a). The radiolabeled, single-strandedDNA and RNA targets are boldly evident in the nonirradiated sample lanes (1, 2,7,8) of the left and right illustrations, respectively. The RNA target ran slightly slower than the DNA relative to xylene cyano1 dye. In all samples that had been UVirradiated (lanes3-6), the target band was greatly reduced and a new isotopic species was resolved as a single, gelretarded band. Its position depended upon which conjugated methylphosphonatespeciesthe irradiated sample contained. With the double conjugate 4 (lanes 5-8), the photoadduct ran slower than that produced with conjugate 5 (lanes 1-4). These observations are consistent with the new species being urea-stable complexes formed of probe and target. One would expect such photoadducts to electrophorese more slowly if tetramethylrhodamine and the tert-amine linker/spacer were attached. In the past, psoralen-conjugatedoligonucleoside methylphosphonates such as control 5 have been shown to efficiently cross-link to complementary single-stranded nucleic acid targets when conjugate/target mixtures are irradiated with nearUV light (Lee et al., 1988a,b;Kean et al., 1988; Bhan and Miller, 1990; Levis, 1993). The rhodamine on conjugate 4 does not prevent cross-linking from occurring. Cross-linking percentages are shown beneath the autoradiographs in Figure 2a, averaged for each pair of duplicatetubes/lanes. Duplicate irradiated tubes differed from each other by a t most 1.8% . Lane 6 on the RNA gel
Thaden and Miller
aI
0 DNA Target
A RNATarget
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
0.3
0.3
90
94
0.3
76
$
1
78
0.3
0
I 0
I1 0 DNATarget
0
*
A RNA Target
I 0
I
I
I
10
20
30
h I
40
I
A
A
I
50
60
Temp ('C)
Figure 2. Denaturing 20% PAGE studies of cross-linkingby an excess of psoralen/rhodamine doubly-conjugated dodecanucleoside methylphosphonate probe, to complementary, singlestranded, 32P-labeledDNA (A)and RNA).( targets. Crosslinking was quantified by scintillation counting of excised gel bands. See the text for details. (a) Effect of the 3'-rhodamine and amino linker (Rhh) and UV irradiation (UVf) on cross-linking at 4 "C. Numbers below gels are cross-linking percentages (mean of duplicate tubes). Lanes: (1,2) Rh-, UV-, (3,4) Rh-, UV+, (5, 6) Rh+, UV+, (7, 8) Rh+, UV-. (b) Effect of temperature on cross-linking (triplicate tubes, duplicate lanes of each; brackets show f1 sd; data from three tubes were discarded due to nuclease degradation of RNA target, one tube each at 20,45, and 55 "C).
was apparently underloaded by about half, but because cross-linkingis a ratio expression, that lane could still be used for quantitation. The mean cross-linking in all irradiated tubes (lanes 3-6, both gels) was 84% (standard deviation,8). Lanes containingnonirradiated samples had negligible isotope (0.3% ) a t the position of the cross-linked complex in the other lanes. The effectiveness of UV-induced cross-linking at 4 "C was sensitive to both variables tested in this experiment. When the backbone ribose sugars in the nucleic acid target were replaced by 2'-deoxyribose (and uracil by thymine), the mean cross-linking increased from 77 % to 92 % (P < 0.00017 ). Differences in DNA and RNA cross-linkingby psoralen-conjugated methylphosphonates have been observed previously.8 When the 3' terminal group of the psoralen-conjugated methylphosphonate probe was changed from a hydroxyl to a group which includes a tetramethylrhodamine, a tertiary amine, and a phosphodiester, the mean crosslinking showed a small but significant increase (83% to 86%, P < 0.004). Therefore, there occurred neither an adverse interaction between the rhodamine and psoralen A two-factor analysis of variance was performed on all data from irradiated samples in order to test for statistical significance of probe effects, target effects, and interaction effects. Kean, J. M., and Miller, P. S., unpublished results.
Rhodamine/Psoralen Conjugated Oligomers
chromophores, nor significant interference by the 3' modification with base-pairing. An interaction between the two variables (probe choice and target choice) was not detected ( P > 0.5); the 3' modification did not detectably prejudice cross-linking in favor of either the RNA or the DNA target during crosslinking at 4 "C. Effect of Temperatureupon Cross-Linking(Probe in Excess). Temperature has proven to be an important variable in sequence-selective cross-linking (e.g. Lee et al., 1988b). Conjugate 4 was incubated a t 11 different temperatures in solutions containing either DNA (6) or RNA (7) radiolabeled targets, and the solutions were irradiated at those temperatures. Cross-linking was calculated and plotted as a function of temperature (Figure 2b). Three different temperature zones could be described: a permissive zone at low temperatures within which cross-linking was efficient, a critical zone spanning 10-15 "C within which cross-linking was extremely sensitive to temperature and inversely related to it; and a prohibitory zone at high temperatures within which crosslinking to RNA was not seen and cross-linking to DNA was not measured. The temperature where half-maximal fell roughly in the middle cross-linking was observed (Tc) of the critical zone. Both the DNA and RNA series showed this quasisigmoidal response to temperature. The advantage enjoyed by DNA in cross-linking with conjugates 4 and 5 at 4 "C (above) was reproduced in this temperature study for conjugate 4 at 4 "C (94% vs 76%; Figure 2a,lanes 543, at the temperature of maximum crosslinking (97% vs 82 % ), and throughout the permissive zone. The most striking differencebetween DNA and RNA crosslinking was the temperature range of the critical zone. The permissive zone for DNA cross-linking persisted to much higher temperatures before the critical zone was reached, a fact best conveyed by the much higher Tc for DNA versus RNA (51 "C vs 29 "C). RNA cross-linking by conjugate 4 exhibited a temperature of half-maximal cross-linking of 29 "C. This is 6 "C higher than any reported to date for RNA cross-linking by a psoralen-derivatized methylphosphonate. As indicated in the 4 "C cross-linking experiment (Figure 2), the presence of the 3' terminal modifications on conjugate 4 may facilitate cross-linking. At higher temperatures, the 3' modification may preferentially aid RNA cross-linking. For both DNA and RNA cross-linking, the initial response to an increase in temperature was a shallow rise in cross-linking, peaking at approximately 20 "C below the Tc.This trend was less obvious with DNA (3% ) than with RNA (6%). Neither target was 100% cross-linked at the temperature of maximal cross-linking. Since the psoralen-derivatized probe was in 50-fold excess over the target, failure to convert 100% of the target to the crosslinked complex was probably not due to incomplete duplex formation during pre-equilibration in the dark, but rather to competing photodegradation of the psoralen moiety (Hearst, 1981). Since the 3' modification is at maximal distance from the psoralen on compound 4, it is likely that the enhanced cross-linking observed at 4 "C (and possibly at higher temperatures with the RNA target) was due to helix stabilization rather than direct action of the rhodamine upon the psoralen. How this occurs is unknown. Possible mechanisms include (a) intercalation or (b) end-stacking by the tetramethylrhodamine, (c) zwitterion formation or (d)reversible ionic cross-linking by the tert-alkylamine in the 3' linker after its quaternization, (e) concerted action by the rhodamine and the amine, and (f) some effect of
BioconJfqate Chem., Vol. 4, No. 5, 1993 381
the amine in ita uncharged, tertiary state. Discussing these in order, conjugation of acridine derivatives at the 3' end of oligonucleotides is known to enhance binding to polynucleotides (HBlBne et al., 1986). The planar, heterocyclicconformation of acridine and other intercalators is a feature also of tetramethylrhodamine in ita ground state. We found no reports in the literature, however, of intercalation by rhodamines, nor did tetramethylrhodamin5-amine or rhodamine B stain the chromatin of cells (not shown). Failure to observe interactions of rhodamine with conventional duplexes does not rule out unique interactions with methylphosphonate heteroduplexes. Melting studies are planned to explore this further. Bauman et al. (1981) saw no difference in the melting temperatures of rhodamine 3' labeled and unlabeled poly(U) when assayed by poly(A)-Sepharose columns, but did see a marginal positive effect of a terminal rhodamine upon hybridization of RNA heteropolymers to complementary DNA-Sepharose. Cardullo et al. (1991) reported an average increase in T, of 4 "C when oligodeoxyribonucleotides of length 6 to 20 were end-linked to rhodamine with an ethyl spacer. Whether by mechanism c or d, a quaternary 3' amine would act to reduce the local charge density of the helix. Concerted stabilization by a 3'acridine derivative and various positively-charged 3' substituents has been described (Asseline et al. 1985).In a recent report, the stability of duplexes of oligonucleoside methylphosphonates and oligodeoxyribonucleotideswas enhanced if the normal oligomer had a 3'-amino group (Gryaznov and Letsinger, 1992). The effect was thought not to be due to the positive charge of the amine. Fluorescence Detection of Cross-Linking (Target in Excess). In the probe-in-excessassays discussed above, unreacted probe was easily visible on gels transilluminated by short-wave UV light (not shown). I t appeared as a red-fluorescent, nonisotopic band running well above the photoadduct band. The 1:l complex, present in less than l/w the amount, was scarcely visible. To increase the yield of the photoadduct, the amount of 2'-deoxy target 6 was increased 320-fold and conjugate 4 was approximately tripled (160 pmol), resulting in a 2:l mole ratio. Solutions were pre-equilibrated and UV-irradiated at 25 "C and then assayed on PAGE gels containing an antifading agent. Figure 3a illustrates autoradiographs (left) and redemission fluorophotographs (right) of parts of four gels from four cross-linking trials. Control lanes from one of the experiments are included: the control in lane 1was masked during UV irradiation; that in lane 2 was irradiated normally, but lacked the DNA 19-mer target; that in lane 3 was irradiated at 55 "C. Lanes 4-4c were loaded with complete reaction solutions which had been UV-irradiated a t 25 "C. All samples were supplemented with a noncomplementary, radiolabeled oligodeoxyribonucleotide11-mer prior to gel loading as an internal control of loading efficiency (band v). Complementary DNA 19-mer 6 was present in all but control lane 2 and appears as a strongly radioactive, nonfluorescent band (iv). The double conjugate 4 was not radiolabeled and is seen only on the fluorophotographs (band ii). Though shorter than oligonucleotide 6, conjugate 4 ran slower because nine of its 11internucleoside linkages were nonionic methylphosphonates. Band ii appears blurred and split in all but lane 1, the only lane loaded with nonirradiated reaction solution. Band ii is markedly attenuated in lanes 4-4c. Those lanes contain a new species (band iii) are discussed below. A fraction of the radioactive material in each lane remained at or near the wells (band i). This phenomenon occurred
392 Bioconjugate Chem., Vol. 4, No. 5, 1993
a)
32PRadioactivity 1 2 3 4 4a4b4c
Thaden and Miller
Red Fluorescence 1 2 3 4 4a4b4c i
mim
ii
-
iv
-9 m
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b) 0 4
812
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,
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,
I
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Figure 3. Cross-linking of a fluorescent, rhodamine/psoralen doubly-conjugatedoligodeoxyribonucleosidemethylphosphonate to excess, 32P-labeled, complementary single-stranded DNA. Photoadducts iii were resolved by denaturing PAGE. See text for details. (a) Autoradiograph (left) and fluorophotograph (right) of portions of four gels from four replicate experiments. Lanes: Abolition of cross-linking by (1)protection from UV light, (2) omission of DNA target, and (3) elevated temperature (55 "C), compared with (4,4a-4c) UV irradiated, DNA-containing solutions treated a t 25 "C.(b) Fluorophotograph (left) and plot (right) illustrating dependence of photo-cross-linkingupon UV fluence. Lane numbers represent minutes of UV exposure. Fraction of fluorescence ( 0 )and of radioactivity (A)found in photoadduct iii is plotted as a function of irradiation time.
regardless of treatment. Its cause is unknown. The wells also appear bright on the fluorophotographs, but upon close inspection, this appears to be due to light having been scattered from or concentrated by the gels' edges. Band iii, the photoadduct, shows up on both the autoradiograph and the fluorophotograph in a position midway between the radioactive band of DNA 19-mer 6 (iv), and the fluorescent b m d of methylphosphonate conjugate 4 (ii). It is strong in lanes 4-4c, the only lanes loaded with complete reaction solutions that were irradiated a t a permissive temperature. On the original films, an extremely faint band was visible at the same position in lanes 1and 3. Some cross-linking may have occurred during gel loading, since room lighting was subdued but not absent. Taken together, these observationsestablish that crosslinking by the doubly-conjugatedoligomer 4 can also occur under the condition of target excess, and that the photoadduct product of its cross-linking (band iii) is a readily detectable, fluorescent species. As in the probein-excessexperiments,cross-linkingstopped short of 100% usage of the less abundant species. Other psoralen photoreactions are known to occur during long-wave UV irradiation besides nucleic acid cycloadduct formation, and these include photodestruction of the psoralen (Hearst, 1981). In lane 2, and in prior studies (discussed in Kean et al., 1988), near-UV irradiation of psoralen-conjugated oligomers generates small amounts of electrophoretically discernable products even in the absence of a nucleic acid
target. For (ae)AMT conjugates such as 4, these were not photoreversible (Lee et al., 1988b). With excess target and pre-equilibration a t 25 "C, nearly all of conjugate 4 would be expected to be duplexed a t the time of irradiation. The blurring and splitting of conjugate 4 (lanes 4-4c) suggest,therefore, that duplex formation does not entirely protect the psoralen moiety from photobreakdown, and that a significant portion of photoproducts in the blurred region were incompetent to covalentlycross-link the DNA strand. This would explain the less-than-100% crosslinking observed under both assay conditions and suggests that duplexed, photodamaged conjugate 4 may act as a competitive inhibitor of cross-linking. In a second target-in-excess cross-linking experiment, solutions contained 240 pmol of oligodeoxyribonucleotide 6 and 120 pmol of conjugate 4. They were UV-irradiated a t 25 "C for periods ranging from 0 to 15 min and then analyzed by PAGE. The fluorophotograph of one of the two gels is reproduced in Figure 3b. The fluorescent (and isotopic) photoadduct was formed in all tubes except the zero-time tube, and could be seen to increase in intensity with longer UV irradiations. The radioactivity and fluorescence of the photoadduct band were quantified by scintillation counting and densitometry respectively, and expressed as fractions of the total signal in the lane. The plot in Figure 3b confirms the pattern of increasing crosslinking seen on the fluorophotograph and shows that radioactivity associated with the photoadduct increases in a concerted fashion. Because the radioactive species was in excess,the fraction complexed was lower. The shape of these curves is similar to those published previously for various psoralen-conjugatedmethylphosphonates (Lee et al., 198813; Bhan and Miller, 1990). Interaction with Mouse L Cells. Fluorescence microscopy of living fibroblasts which had been grown for 9 h in medium containing conjugate 4 showed strong rhodamine fluorescence associated with the cells (Figure 4). Treated cells had a mean brightness 2.5-fold higher than that due to back-scatter and autofluorescence in untreated cells and had a maximum brightness 7.4-fold that of the brightest point in untreated cells. The cells pictured in Figure 4 exemplify the predominant fluorescence distribution pattern seen with both conjugate 4 and conjugate 3 under a wide range of treatment times and concentrations. The most intense signal was found in points or speckles in the cytoplasm, most of which were less than 1pm in diameter. In the majority of cells, the bright points were particularly concentrated and coalesced next to, over, or under the nucleus. This pattern differs markedly from the generalized cytoplasmic fluorescence gotten when the free rhodamine dye, tetramethylrhodamin5-amine, was administered to L cells (not shown). A number of laboratories have reported punctate, perinuclear patterns of fluorescence in cells in culture treated with a variety of fluorescent oligonucleotides and oligonucleotideanalogues (Jaroszewski and Cohen, 1991; Bennett et al., 1992;Iversen et al., 1992;Marti et al., 1992; Shoji et al., 1991; Thierry and Dritschilo, 1992). Some nuclear localization has also been noted. The juxtanuclear pattern of spots is generally attributed to confinement of the reagents to vesicles, particularly endosomes, and cycling of the endosomes between the plasma membrane and the Golgi area. Endosomal containment could potentially limit the bioavailabilityof antisense and antigene reagents a t their presumed sites of action. While the vesicular pattern is strikingly bright due to local concentration, a significant amount of the total cellular fluorescence was distributed diffusely over the
Rhodamine/Psoralen Conjugated Oligomers
Bioconjugate Chem., Vol. 4, No. 5, 1993 393
Liposomally encapsulated, phosphorothioate-capped oligonucleotides eventually migrated to the nucleus also (Thierry and Dritschilo, 1992). In this article, we have detailed the synthesis of a conjugate formed of three well-characterized molecules: oligonucleoside methylphosphonate, tetramethylrhodamine, and 4’- [N-(aminoethy1)aminomethyll-4,5’,8trimethylpsoralen. We have demonstrated that the linkage between the methylphosphonate and the rhodamine is stable in serum-containing medium. The linkage to psoralen has previously been shown to meet that test. We have demonstrated the capacity of the conjugate to crosslink to single-stranded, complementary oligoribo- and oligodeoxyribonucleotides in vitro. Finally, we have demonstrated that the conjugate can be taken up by fibroblastic cells growing in monolayer culture and have given some indication of its distribution in these cells. The stability, fluorescence, and cell penetrability of the psoralen/rhodamine doubly-conjugated oligonucleoside methylphosphonate 4 are properties required if one is to use it for microscopic studies of antisense behavior in living cells and tissues. The photoaffinity it shows for complementary single-stranded DNA and RNA in vitro affords an opportunity to directly assay for antisense interaction of an antisense reagent with messenger RNA in living cells. ACKNOWLEDGMENT
~~
Figure 4. Cellular distribution of a fluorescent, psoralenl rhodamine doubly-conjugated oligodeoxyribonucleosidemethylphosphonate. Color composite image formed by alignment of a Nomarski differential interference contrast image (blue-green) and an epifluorescence image (red) of live mouse L929 fibroblasts cultured for 9 h in medium containing 5 pM of the oligomer conjugate (see the text for details). Length of bar is 10 pm. Inset: Black-and-white epifluorescence image of the same cells, but with fluorescence intensity displayed on a logarithmic scale to emphasize less intensely fluorescent areas.
entire cell area. This is apparent if one displays the same cells with fluorescence brightness displayed on a logarithmic scale in order to de-emphasize areas of extraordinary intensity (Figure 4, inset). Finally, an “outline” effect was sometimes also seen, where part or all of a cell boundary appeared bright. This is a feature observed when fluorescent probes react with elements of the plasma membrane (Willingham and Pastan, 1985). Also characteristic of a membranal distribution is a vesicular, cytoplasmic pattern focused in the Golgi area, due to membrane recycling, and a weak fluorescence over the entire cell due to overlying and underlying cell membrane. The distribution illustrated in Figure 4 differs from a purely membranal one in that the outline effect is not very striking and not visible on every cell. Diffuse brightness over the cell area could be due to Rayleigh scattering of the intense emission from vesicularized probe, or it could be due to presence of conjugate 4 in the cytosol. If conjugate 4 was free in the cytosol, then its behavior in these mouse fibroblasts is not as one might predict from studies where fluoresceinated methylphosphonates and rhodamine-labeled oligodeoxyribonucleotides were microinjected into the cytoplasm of human and mouse fibroblasts respectively (Chin et al., 1990; Leonetti et al., 1991). These conjugates were reported to become rapidly sequestered in the nucleus.
Financial support for this work was provided through grants from the National Cancer Institute (CA42762),the National Institutes of Health (ES03841 and CA09110), and the Department of Energy (DEFG02-88ER60636). We gratefullyacknowledgethe expert advice of J. M. Kean, who also generously provided us with oligomers 5-7. S. A. Lesko and D. E. Callahan provided invaluable training and advice on microscopy and image processing. (ae)AMT was synthesized by P. Bhan. HPLC with diode-array detection was done with the assistance of S. D. Morrow. LITERATURE CITED Agrawal, S., and Goodchild, J. (1987) Oligonucleoside methylphosphonates: Synthesis and enzymatic degradation. Tetrahedron Lett. 28, 3539-3542. Aikens, R. S., Agard, D. A., and Sedat, J. W. (1989) Solid-state imagers for microscopy. Meth. Cell Biol. 29, 291-313. Akhtar,S.,Kole, R., and Juliano, R. L. (1991)Stability of antisense DNA oligonucleotide analogues in cellular extracts and sera. Life Sci. 49, 1793-1801. Asseline, U., Nguyen, T. T., and Hblhne, C. (1985) Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences. J. Biol. Chem. 260,8936-41. Bauman, J. G. J., Wiegant, J., and Van Duijn, P. (1981) Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model. J. Histochem. Cytochem. 29,227237.
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