An Alternative Approach to Iterative Solid-Phase Synthesis - American

Feb 15, 1997 - Departments of Oligonucleotide Chemistry and Cell Biology, Ribozyme Pharmaceuticals, Inc., 2950 Wilderness. Place, Boulder, Colorado ...
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Bioconjugate Chem. 1997, 8, 204−212

204

Post-synthetically Ligated Ribozymes: An Alternative Approach to Iterative Solid-Phase Synthesis Laurent Bellon,*,† Christopher T. Workman,† Thale C. Jarvis,‡ and Francine E. Wincott† Departments of Oligonucleotide Chemistry and Cell Biology, Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, Colorado 80301. Received November 25, 1996X

To improve the overall yield of ribozyme synthesis, a convergent approach, based on the post-synthetic formation of an amino linker between two half-ribozymes was investigated. Borane‚pyridine-mediated reductive amination of 3′-phosphoglycaldehyde-5′-half-ribozymes with 5′-aminohexyl-3′-half-ribozymes generated the corresponding amino-linked ribozymes in yields >77% on different scales. The investigation of a variety of reducing agents is discussed together with a kinetic analysis of the selected coupling reaction. These post-synthetically ligated ribozymes exhibited slightly reduced in vitro catalytic activity and cell efficacy.

INTRODUCTION

MATERIALS AND METHODS

Ribozymes are RNA enzymes (Cech, 1993; Symons, 1994) that can be designed to cleave other RNA molecules. trans-Cleaving hammerhead ribozymes show great promise as therapeutic agents due to their inherent catalytic activity combined with highly specific binding to a chosen target RNA (Christoffersen and Marr, 1995). Improvements in the chemical synthesis of RNA (Wincott et al., 1995; Scaringe et al., 1990) have led to the sitespecific introduction of various chemical modifications into hammerhead ribozymes providing nuclease resistance (Usman and Cedergren, 1992; Yang et al., 1992) and enhanced catalytic activity (Beigelman et al., 1995; Burgin et al., 1996). Because of the iterative nature of solid-phase oligoribonucleotide synthesis, only modest chemical yields of a 37-mer ribozyme can be achieved. Moreover, the necessary reversed-phase and anionexchange purification steps further diminish this yield due to the inherent difficulty in separating the full-length ribozyme from the failures. As part of an ongoing effort to overcome these limitations, our group has designed an alternative approach (Bellon et al., 1996) in which two half-ribozymes are synthesized using known solid-phase methodologies. These halves contain complementary chemical functionalities that allow post-synthetic chemical ligation through a covalent linkage. Due to their lengths and the iterative synthesis process, the halfribozymes are obtained in greater yield as compared to a full-length ribozyme. Therefore, this half-ribozyme approach has a theoretical advantage over the purely solid-phase procedure, with respect to yields, provided that the post-synthetic chemical ligation proceeds efficiently. We describe herein the design, synthesis, and reductive amination coupling of half-ribozymes, on a pilot scale, together with a kinetic analysis of the conjugation reaction. The optimized conditions were then applied to a larger scale synthesis (50 µmol), allowing a direct comparison between the half-ribozyme strategy and classical recurrent ribozyme synthesis. Catalytic activity and cell culture efficacy of the amino-linked ribozymes are also discussed.

General Methods. Glyceryl-CPG and aminohexyl linker phosphoramidite were purchased from GlenResearch, Sterling, VA. Sodium cyanoborohydride, sodium borohydride, sodium triacetoxyborohydride, borane‚pyridine, borane dimethylamine, and amberlyst A-26 borohydride were obtained from Aldrich Chemical Co., Milwaukee, WI. Sep-Pak Plus (C18) purification cartridges were obtained from Waters Corp., Milford, MA. Oxidative Cleavage of the 3′-Phosphoglyceryl-5′half-ribozyme. A typical procedure is represented with half-ribozyme 4. Half-ribozyme 4 (100 µL, 50 nmol) was oxidized over 30 min with 10 equiv of aqueous NaIO4 (1 µL, 500 mM). Small-Scale Desalting of the Ribozymes after Periodate Oxidation or Anion-Exchange HPLC. A typical procedure is represented with half-ribozyme 8. The crude oxidized 3′-phosphoglycaldehyde-5′-halfribozyme 8 was applied to a Waters Sep-Pak Plus (C18) cartridge conditioned with CH3CN/MeOH/H2O (1:1:1, 10 mL) and SuperQ H2O (20 mL). Following sample application, the cartridge was washed with SuperQ H2O (10 mL) to remove formaldehyde and excess sodium periodate or excess sodium chloride in the case of anion-exchangepurified samples. Product was then eluted from the column with CH3CN/MeOH/H2O (1:1:1, 10 mL) and dried under reduced pressure. Reductive Amination Ligation of 5′- and 3′-HalfRibozymes. A typical procedure is represented by the synthesis of the amino-linked ribozyme 9. 3′-Phosphoglycaldehyde-5′-half-ribozyme 8 (10 µL, 500 µM) and 5′-aminohexyl-3′-half-ribozyme 5 (10 µL, 500 µM) were mixed in a 1.5 mL Eppendorf tube. Sodium N2-acetamido-2-iminodiacetate (ADA) (20 µL of 200 mM, pH 6.2) was then added (125 µM half-ribozyme final concentration). Reductive amination coupling was initiated with borane‚pyridine in EtOH (1 µL, 160 mM, 30 equiv). Reaction was sampled (0.5 µL, 60 pmol) every few hours and diluted in water (200 µL, 180 µL injected) for HPLC analysis. Analytical Anion-Exchange HPLC. All analyticals were run on a Hewlett-Packard 1090 HPLC using a Dionex NucleoPac PA-100 column, 4 × 250 mm. Perchlorate buffers (buffer A ) 10 mM NaClO4/1 mM Tris‚HCl; buffer B ) 300 mM NaClO4/1 mM Tris‚HCl, both pH 9.3) and column heating to 50 °C were standard conditions. Analyticals were run with 30-60 pmol of ribozyme injected in a volume of 180 µL. Half-ribozymes

* Author to whom correspondence should be addressed (email [email protected]). † Department of Oligonucleotide Chemistry. ‡ Department of Cell Biology. X Abstract published in Advance ACS Abstracts, February 15, 1997.

S1043-1802(97)00011-6 CCC: $14.00

© 1997 American Chemical Society

Post-synthetically Ligated Ribozymes

were analyzed on a 30-60% B gradient over 12 min. Oxidized 3′-phosphoglycaldehyde-5′-half-ribozymes were run at 80 °C on a 45-75% B gradient over 12 min. Reductive amination reactions were analyzed on a 4070% B gradient over 12 min. Small-Scale Purification of the Amino-Linked Ribozymes. Crude samples (5 mL) were injected onto a Dionex NucleoPac PA-100, 22 × 250 mm (90 mL), column equilibrated with buffer A (buffer A ) 20 mM NaCl/10% EtOH/1 mM Tris‚HCl; buffer B ) 1 M NaCl/ 10% EtOH/1 mM Tris‚HCl, both pH 9.3). The linked ribozymes were purified at elevated temperatures since heat was necessary to melt the hybrid formed between the unreacted halves. A 55-70% B gradient was applied over 60 min. A 10 mL/min flow rate was used, and fractions were collected every minute. Fractions containing full-length product >80% by peak area were pooled and desalted using the method described for 8. Large-Scale Reversed-Phase HPLC Purification of the 5′-Half-Ribozyme 4 and Full-Length Control RPI.3718. The crude material from a large-scale tritylon (50 µmol) synthesis was applied to a Pharmacia Source 15RP 16/10 column equilibrated in 100% buffer A (buffer A ) 1 M NaCl/5 mM Tris‚HCl, pH 9.0; buffer B ) 60% EtOH/5 mM Tris‚HCl, pH 9.0) on a FPLC system (Pharmacia Biotech). A gradient from 0 to 40% B in 4 column volumes (CVs), from 40 to 42% B in 5 CVs, and then from 42 to 100% B in 3 CVs was applied at a flow rate of 10 mL/min (300 cm/h). Fractions containing over 60% full-length material by HPLC were pooled and subjected to manual detritylation with HCl. The solution was acidified to pH 2 (20-40 mM HCl) with 1 M HCl for 15 min. The pH of the solution was then adjusted to pH 7 with Tris base (1 M Tris base, pH 11-12). This material was analyzed by HPLC at >70% full-length by area. Large-Scale Anion-Exchange HPLC Purification of the Half-Ribozymes 4 and 5, Full-Length Control RPI.3718, and Amino-Linked Ribozyme 9. The reversed-phase purified, detritylated material was applied to a Pharmacia Source 15Q 26/10 (50 mL) anion-exchange column on a Pharmacia FPLC system. A NaCl gradient (buffer A ) 20 mM NaCl/5 mM Tris‚HCl, pH 9; buffer B ) 1 M NaCl/5 mM Tris‚HCl, pH 9) was applied at a flow rate of 10 mL/min. The half-ribozymes were purified using a 30-50% B gradient in 40 CVs. For the fulllength control, RPI.3718, a gradient from 40 to 60% B in 40 CVs was used. The linked ribozyme 9 was purified at 55 °C with a gradient from 60 to 80% B in 20 CVs. The fractions containing over 80% full-length material were pooled. This material was analyzed by HPLC at >85% full-length by area. Large-Scale Desalting of the 3′-Phosphoglycaldehyde-5′-half-ribozyme 8 and of the AnionExchange-Purified Ribozymes. The ribozyme solutions were applied to a 1.6 × 10 cm (20 mL) bed of Bondapak C18 125 Å (37-55 µM) packed in a Pharmacia HR 16/10 column pre-equilibrated in water. Application and desalting were run at a flow rate of 10 mL/min. Ribozyme was cleared from the column with a step gradient to 30% EtOH in water. All UV-absorbing fractions were pooled and dried under reduced pressure. Electrospray Mass Spectrometry. Desalting was performed using a modified ammonium acetate precipitation procedure (Stults and Marsters, 1991). Ribozyme (10 nmol) was suspended in SuperQ water (30 µL). NH4OAc (50 µL, 5 M, pH 5.6) was added and the solution vortexed. After 10 min, absolute EtOH (600 µL) was added and the ribozyme solution vortexed and placed in the -70 °C freezer overnight. Samples were centrifuged

Bioconjugate Chem., Vol. 8, No. 2, 1997 205

(14 000 rpm, 30 min), and supernatant was carefully removed. The pellets were treated twice more with this procedure to ensure efficient ammonium exchange. ESMS was performed on a Fison Instruments VG QuattroSG quadrapole mass spectrometer. Desalted and ammonium-exchanged samples were suspended in deionized water (1 nmol/µL). The ribozyme solution (1 µL) was added to an acetonitrile (ACN) solution [17 µL, 80% ACN/ 2.5 mM 1,2 diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA)/0.1%TEA]. The ribozyme/ACN solution was injected through a fused silica loop (10 µL) at 4 µL/min. The instrument was run in ES(-) mode (cone voltage ) 42 V) on a mass range of 400-1500. Ribozyme Catalytic Activity Assay. RPI.3718, amino-linked ribozyme 9, its inactive version, and 5′-32Pend-labeled substrate 3 were heated separately in reaction buffer (50 mM Tris‚HCl, pH 8.0; 40 mM MgCl2) to 95 °C for 2 min, quenched on ice, and equilibrated to the final reaction temperature (37 °C) prior to the start of the reactions. Reactions were carried out in enzyme excess and were started by mixing ∼1 nM substrate with 500 nM ribozyme in a final volume of 50 µL. Aliquots of 5 µL were removed at 0.5, 1, 2.5, 5, 10, 15, 20, 30, and 60 min, quenched in formamide loading buffer, and loaded onto 15% polyacrylamide/7 M urea gels. The fraction of substrate and product present at each time point was determined by quantitation of scanned images from a Molecular Dynamics PhosphorImager. Ribozyme cleavage rates were calculated from plots of the fraction of substrate remaining vs time using a double-exponential curve fit (Kaleidagraph, Synergy Software) (Burgin et al., 1996). For RPI.3718, the fast portion of the curve represented 54% of the total reaction; therefore, the observed cleavage rate (kobs) was taken from fits of the first exponential. For amino-linked ribozyme 9, the slow portion of the curve represented 93% of the total reaction; therefore, the observed cleavage rate (kobs) was taken from fits of the second exponential. Smooth Muscle Cell Proliferation Assay. Rat aortic smooth muscle cells (RASMC) were isolated and propagated as described (Jarvis et al., 1996a). Cell proliferation assays were performed according to a modification of the method of Jarvis et al. (1996a). Briefly, cells were set at a density of 5000 cells per well in a 24-well tissue culture plate and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS for 24 h at 37 °C in a 5% CO2 atmosphere. Cells were then washed twice in Dulbecco’s phosphate-buffered saline (DPBS) and serum starved in DMEM containing 0.5% fetal bovine serum (FBS) for 48 h to induce a quiescent state. Cells were then washed twice with DPBS and treated with 0.5 mL per well of 100 nM ribozyme complexed with 7.2 µg/mL LipofectAMINE (GIBCO-BRL) for 1.5 h at 37 °C. Ribozyme/lipid complexes were then removed, and the cells were washed twice with DPBS and incubated in DMEM containing 0.25% FBS for an additional 6 h. Cells were then stimulated to proliferate by addition of 10% FBS. Cell proliferation was measured by incorporation of BrdU as described (Jarvis et al., 1996a). RESULTS AND DISCUSSION

Synthetic Strategy. A realistic averaged stepwise chemical yield (ASWY) of 96.5% can be routinely obtained for ribozyme synthesis as determined by the ratio (µmol of FLR/µmol scale)1/n × 100, where µmol of FLR is the amount of full-length ribozyme in the crude mixture, µmol scale is the scale of synthesis and n is the number of synthesis cycles. This half-ribozyme approach has a theoretical advantage over the iterative procedure with

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Figure 2. Structures of RPI.3718 1, RPI.3718S 2, and the RNA substrate 3. Figure 1. Theoretical chemical yield comparison between halfribozyme approach and iterative ribozyme synthesis. Ligated ribozyme synthesis yields are calculated as follows: (49% × ligation yield), where 49% is the 21-mer half-ribozyme synthesis yield as determined from a 96.5% ASWY; or (49% × ligation yield) × 80%, where 80% is the recovery yield of the additional purification step for the convergent route. The selected breakeven point for the two strategies corresponds to 71%.

respect to yields. A 96.5% ASWY correlates in 28% theoretical yield for a 37-mer ribozyme, 49% theoretical yield for a 21-mer half-ribozyme, and 56% theoretical yield for a 17-mer half-ribozyme. A critical requirement is the chemical ligation yield of the two halves to form the full-length ribozyme (Figure 1). We have determined that a minimum coupling yield of 57% is theoretically sufficient to compete with iterative synthesis (49% × 57% ) 28%). However, the convergent route requires an additional purification step to separate the chemically ligated full-length ribozyme from the unreacted halves. Therefore, we have set the threshold that ensures the advantage of this segmented approach over iterative synthesis at 71% half-ribozyme coupling yield (Figure 1). It is clear from Figure 1 that any improvement in the ligation efficiency that raises it above 71% will provide a greater amount of full-length ribozyme. Ideally, quantitative ligation efficiency would lead to 39% FLR, to be compared with 28% for the iterative approach (Figure 1). Design of Half-Ribozymes. An important requirement for this strategy to be successful is that the site of chemical ligation must not interfere with the ribozyme core to ensure that full catalytic activity is retained. It has been previously shown that the stem II or loop II of the hammerhead ribozyme is not essential for catalytic activity (Bellon et al., 1996; Tuschl et al., 1993; Benseler et al., 1993; Beigelman et al., 1994; Hendry et al., 1994). Similarly to our previous work (Bellon et al., 1996), the standard GAAA tetraloop II and the stem II region of the generic, nuclease stable ribozyme motif (Beigelman et al., 1995; Burgin et al., 1996), as represented by RPI.3718 (Jarvis et al., 1996a) 1 (Figure 2), were modified to accommodate post-synthetic chemical coupling (Figure 3). Chemical ligation or conjugation of oligonucleotides can be greatly enhanced by the presence of a template (Gryaznov and Letsinger, 1993; Herrlein et al., 1995). Therefore, we assumed that successful coupling reaction

Figure 3. Structures of the 5 base-pair and 4 base-pair stem II half-ribozymes 4, 5, 12, 15, and 16 and 6 and 7, respectively.

of the two half-ribozymes relied upon formation of the stem II bringing the two chemical moieties within close proximity to one another. Free energies predicting duplex stability of the suitable 5′-3′ (4.5 or 6.7) and undesired 5′-5′ (4.4 or 6.6) or 3′-3′ (5.5 or 7.7) duplexes were calculated (Freier et al., 1986) with the standard self-complementarity ggcc stem II (as represented in 6 and 7, Figure 3) or with a modified gcacc five base-pair stem II (as represented in 4 and 5, Figure 3) (Table 1). The ∆G° values were clearly in favor of the 5 base pair stem II ensuring the formation of the correct duplex 4.5 by -3.3 kcal mol-1 over the other possible duplexes 4.4 or 5.5. Furthermore, a uridilyl residue was introduced between the 3′-phosphoglyceryl moiety and the stem II in the 5′-half-ribozyme 4 (Figure 3) to allow spatial bridging between the primary amine of 5 and the carbonyl

Table 1. Free Energies ∆G° Calculation Based on Nearest-Neighbor Model, 37 °C, 1 M NaCl or 10 mM Mg2+ 5 bp (gcacc)

stem II duplex ∆G° (kcal mol-1)

4.5 (5′-3′) -11.7

4.4 (5′-5′) -8.4

4 bp (ggcc) 5.5 (3′-3′) -8.4

6.7 (5′-3′) -10.4

6.6 (5′-5′) -10.4

7.7 (3′-3′) -10.4

Post-synthetically Ligated Ribozymes

Bioconjugate Chem., Vol. 8, No. 2, 1997 207

Figure 4. (A) NaIO4-mediated oxidative cleavage of the 3′phosphoglyceryl-5′-half-ribozyme 4 to the 3′-phosphoglycaldehyde 8. (B) Anion-exchange HPLC analysis of the oxidative cleavage reaction after 0, 10, and 30 min.

functionality of the oxidized 4. Indeed, preliminary work on morpholino-linked ribozymes (Bellon et al., 1996) indicated that a single atom difference between an aminohexyl linker and an aminoethylene glycol linker had a tremendous influence on the extent of product formation (data not shown). Reductive Amination Coupling of Half-Ribozymes. We selected a linear amino linkage to covalently bridge the 5′- and 3′-half-ribozyme since reductive amination chemistry has been applied effectively to a wide range of oligonucleotide substrates under aqueous conditions (Goodchild, 1990; Morvan et al., 1996; Deschamp and Sonveaux, 1995; Harambilis et al., 1994; Lemaitre et al., 1987). The previously studied morpholino linkage (Bellon et al., 1996) was not chosen here because of long reaction time together with the formation of two linked products. Half-ribozymes 4 and 5, derived from RPI.3718, were synthesized on a 2.5 µmol scale on glyceryl-controlled pore glass (Urata and Akagi, 1993) and on an inverted abasic polystyrene (Jarvis et al., 1996b), respectively, and then purified according to standard methods (Wincott et al., 1995). The 3′-phosphoglyceryl-5′-half-ribozyme 4 (125 µM aqueous solution) was subjected to oxidative cleavage with 10 molar equiv of a 100 mM aqueous solution of sodium periodate (Figure 4A). Complete conversion of 4 to the 3′-phosphoglycaldehyde-5′-half-ribozyme 8 could be observed within 30 min (Figure 4B). Since the periodate-mediated oxidation of the 3′-phosphoglyceryl 4 generates 1 molar equiv of highly reactive formaldehyde, 8 was eluted from the reaction mixture on a SepPak C18 cartridge (Waters Corp.). Half-ribozymes 8 (125 µM) and 5 (500 µM) were then reacted with 30 molar equiv of aqueous NaBH3CN (Borch et al., 1971) (500 mM) in ADA buffer (100 mM), pH 6.0 (Figure 5A). Unlike the morpholino ribozyme (Bellon et al., 1996), after 7 days of reaction, a substantial amount of unreacted 3′-phosphoglycaldehyde 8 could be observed (Figure 5B). After HPLC purification, 9 was identified as the desired amino-

Figure 5. (A) NaBH3CN-mediated coupling of the amino-linked ribozymes 9 and 10. (B) Anion-exchange HPLC analysis of the reductive amination of 8 with 5 after 0 and 150 h.

linked ribozyme on the basis of ES-MS analysis (calcd 11928.6, found 11929.0). Product 10 was assigned as a cyanoborane adduct in accordance with our previous data (Bellon et al., 1996). As a result of the incomplete reaction of the half-ribozymes, in addition to the undesired formation of the adduct 10, the amino-linked ribozyme, 9, was obtained in only 50.2% yield (Table 2). Since the necessary 71% coupling efficiency (Figure 1) was not obtained, we investigated other reducing agents to suppress the formation of the adduct as in 10 yet allow faster and higher yielding synthesis of 9. We assumed that the borane adduct formation could be prevented with the use of borane reducing reagents already complexed with an amine (Pelter et al., 1988). Such amine-borane complexes have reducing properties similar to those of hydroborates (Pelter et al., 1988), which are known to be rather selective for Schiff base reduction. We investigated the use of borane‚pyridine (Pelter et al., 1984; Bomann et al., 1995; Moorman, 1993) and borane‚dimethylamine (Billman and McDowell, 1961) complexes together with other types of hydroborates such as sodium borohydride (Abdel-Magid et al., 1994), sodium triacetoxyborohydride (Abdel-Magid et al., 1990; Hart

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Table 2. Reductive Amination of 8 (125 µM) with 5 (500 µM) in ADA (100 mM, pH 6.0) with 30 Molar Equiv of Reducing Agent for 48 h at Room Temperaturea reducing agent 9 (%) 10 (%) c

NaBH3CNb

NaBH(OAc)3c

BH4‚Amberlyst A-26d

NaBH4c

BH3‚Pyre

BH3‚HNMe2c

50.2 14.5

NRf

NR NR

NR NR

81.2 NR

4.4 NR

NR

a Yields are expressed as µmol ratio × 100 based on the disappearance of the limiting reagent 8. b 500 mM in H O, 7 days of reaction. 2 100 mM in H2O. d2.5 mmol equiv of BH4- g-1 of resin. e 80 mM in EtOH. f NR, no significant reaction after 48 h.

Figure 6. Anion-exchange HPLC analysis of the borane‚pyridine-mediated coupling of the amino-linked ribozyme 9 after 0 and 48 h of reaction.

and Leroy, 1995), or borohydride exchange resins (Yoon et al., 1993; Gibson and Bailey, 1977). Half-ribozymes 8 (125 µM) and 5 (500 µM) were reacted in ADA buffer (100 mM), pH 6.0, in the presence of 30 molar equiv of the reducing agent for 48 h (Table 2). As seen from Table 2, borane‚pyridine complex produced a very high yield of the amino-linked ribozyme 9 without the concomitant formation of a second ligated product. A small amount of unreacted 5′-half-ribozyme was still observed after 48 h of reaction (Figure 6). Following anion-exchange purification, this material was subjected to ES-MS to determine whether 8 could have been reduced (Andrews and Crawford, 1980) into the corresponding 3′-phosphoglycol, 11 (Urata and Akagi, 1993), thereby competing

with the reductive amination. The mass analysis (calcd 6652.2, found 6653.0) was in agreement with the molecular weight of alcohol 11, although 8 (calcd 6650.2) and 11 (calcd 6652.2) only differ by 2 atomic mass units. The fact that 11 did not react with the 5′-aminohexyl-3′-halfribozyme 5 when subjected to the same reductive amination conditions (data not shown) provided supportive evidence regarding the 3′-phosphoglycol nature of 11. Reducing the molar equivalent of borane‚pyridine to 5 equiv lengthened considerably the reaction time (over 5 days) without preventing the formation of 11. Adding the 30 molar equiv in two portions at a 12 h interval did not change the scope of the reaction (data not shown). Kinetic Analysis of the Borane‚Pyridine-Mediated Half-Ribozyme Coupling. To prepare for the large-scale (50 µmol) comparison of the half-ribozyme approach vs recurrent synthesis, we felt it was important to fine-tune reaction conditions giving rise to the aminolinked ribozyme 9. In particular, optimal reaction time and concentration of the halves were investigated. Coupling reactions were set in ADA buffer (100 mM), pH 6.0, in the presence of 30 molar equiv of BH3‚Pyr (800 mM in ethanol) with 1.25 mM, 125 µM, or 12.5 µM of the 3′phosphoglycaldehyde-5′-half-ribozyme 8 and the 5′aminohexyl-3′-half ribozyme 5 (1:1 stoichiometry). Anionexchange HPLC monitoring of the coupling reaction allowed us to plot the formation of the amino-linked ribozyme, 9 (expressed in percent yield), vs time (Figure 7A). The 125 and 12.5 µM reactions reached the same 72% coupling yield after 24 and 150 h, respectively, whereas a final 56% coupling efficiency could be obtained after only 5 h with the high 1.25 mM concentration (Figure 7A). Extending the reaction time up to 150 h for the three concentrations did not have any significant impact on the amount of product formed. One possible

Figure 7. (A) Kinetic plots of the formation of 9 over time using 1.25 mM, 125 µM, or 12.5 µM stoichiometric amounts of half-ribozymes 5 and 8. (B) Kinetic plot of the rescue reaction using 125 µM 5 and 2 × 125 µM 8: (1) formation of 9; (2) consumption of 5.

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Figure 8. (A) Anion-exchange HPLC analysis of the NaIO4-mediated oxidative cleavage of the thiolated 3′-phosphoglyceryl-halfribozyme 12 to the 3′-phosphoglycaldehyde 13, after 0 and 30 min reaction times. (B) Anion-exchange HPLC analysis of the borane‚pyridine-mediated coupling of the amino-linked ribozyme, 14, after 0 and 48 h of reaction and after purification. Table 3. Initial Velocities of the Reductive Amination Cross-Coupling Reaction half-ribozyme 5 and 8 concentration 1.25 mM kobs (h-1)

0.611

125 µM 0.290

Table 4. Cleavage Rate of the Substrate 3 by the “Active” Ribozyme 9, Its “Inactive” Analog, and RPI.3718Sa ribozyme

12.5 µM 0.086

explanation for this “plateau” effect is that the 3′phosphoglycaldehyde-5′-half-ribozyme 8 was competitively reduced into the corresponding alcohol (Andrews and Crawford, 1980) during the reductive amination with 5. To further validate this hypothesis, we designed a “rescue” experiment in which another equivalent of halfribozyme 8 was added to the 125 µM reaction after 24 h. As predicted, this procedure increased the final coupling yield to 93.6% after 48 h (Figure 7B). Another “rescue” experiment in which a molar equivalent of 5 was added to the 125 µM reaction after 24 h did not have any effect on the formation of the product 9 (data not shown), corroborating the fact that 8 was consumed in a side reaction. Since these “rescue” experiments did not use the desired 1:1 stoichiometry, the single mixing of an equimolar amount of half-ribozymes (125 µM) together with a 24 h reaction time was selected for the 50 µmol large-scale comparison. The experimental time points (Figure 7A) were treated (Burgin et al., 1996) using a double-exponential curve algorithm (KaleidaGraph). Kinetic analyses of the initial velocities of the reactions revealed a direct relationship between half-ribozyme concentration and the initial rate constant (Table 3). The lower yield (72%) obtained in these kinetic experiments was attributed to the 1:1 stoichiometry since it is known that reductive amination works optimally in the presence of a 5-fold excess of the amine residue (Borch et al., 1971). Catalytic Activity of the Amino-Linked Ribozyme 9 and Its “Inactive” Counterpart. Once the aminolinked ribozyme 9 was synthesized and characterized, it was critical to ascertain the effect of this chemical linkage on the ribozyme activity. “Active” amino-linked ribozyme 9, its “inactive” counterpart containing two mutations in the catalytic core that abolish cleavage activity (Beigelman et al., 1995; Jarvis et al., 1996) (see 4 and 5, Figure 3), and the control RPI.3718 were assayed under singleturnover conditions for their cleavage rate on short substrate 3 (Figure 2). The amino-linked ribozyme 9 was approximately 10 times slower than RPI.3718 (Table 4), confirming that one can extensively modify the stem II/ loop II region without dramatically affecting cleavage activity. As expected, the inactive amino-linked ribozyme completely lacked detectable catalytic activity.

kobsb (min-1)

“active” 9

“inactive” 9

RPI.3718

0.012

40 µmol g-1) CPG solid supports are known (Wright et al., 1993) to lose the mechanical and fluidic properties suitable for large-scale

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Bioconjugate Chem., Vol. 8, No. 2, 1997 211

anced by the necessity of using a 3′-phosphoglyceryl-5′half-ribozyme that cannot be synthesized in satisfactory yields. ACKNOWLEDGMENT

We thank Susan Grimm, Victor Mokler, and Lara Maloney for the synthesis and purification of the different oligoribonucleotides, James McSwiggen for free energy calculations, Carolyn Gonzalez for catalytic activity assay, Alex Burgin for fruitful kinetic discussions, and Nassim Usman for continuous support. LITERATURE CITED Figure 11. (A) Chemical yields of RPI.3718, half-ribozymes 4 and 5 after synthesis, reversed-phase and anion-exchange purification. (B) Chemical yield of the amino-linked ribozyme 9 after coupling and anion-exchange purification.

synthesis. Therefore, we functionalized highly crosslinked aminomethyl polystyrene solid support (McCollum and Andrus, 1991) with 1-O-dimethoxytritylglycerol according to a known procedure (Urata and Akagi, 1993). Under these conditions, glyceryl polystyrene solid support was obtained with a 11.6 µmol g-1 loading yield. Unfortunately, 4 synthesized from this resin was not obtained in any better yield as compared to CPG. This led us to believe that the electron-withdrawing acetoxy group (Urata and Akagi, 1993) adjacent to the dimethoxytrityl residue diminished the reactivity of the primary hydroxyl group generated after detritylation. It was obvious that since 4 was obtained with a lower chemical yield (6.88 µmol) than the full-length control RPI.3718 (8.88 µmol, Figure 11), the coupling of the two halves could not generate more material than the iterative synthesis. However, the coupling study was completed to evaluate the scale-up of the pilot reactions (Figure 11B). 3′-Phosphoglyceryl-half-ribozyme 4 (1305 AU260 nm, 6.88 µmol) was oxidized as above into the 3′phosphoglycaldehyde 8, which was desalted on reversedphase HPLC to remove excess NaIO4 and formaldehyde. The aldehyde 8 was coupled in ADA buffer (100 mM, pH 6.0) with a stoichiometric amount of 5′-aminohexyl-halfribozyme 5 (994 AU260 nm, 6.88 µmol, Figure 11B, final half-ribozymes concentration ) 116 µM) in the presence of 30 molar equiv of BH3‚Pyr (80 mM in ethanol) for 24 h. As expected, the amino-linked ribozyme 9 was obtained cleanly in 77.6% coupling yield, well above the 71% threshold (Figure 1). After a final anion-exchange HPLC purification step to remove the unreacted 5′- and 3′halves, 1344 AU260 nm (4.04 µmol) of 9 exhibiting a 97.4% HPLC spectrophotometric purity at 260 nm was isolated (Figure 11B). This corresponds to a 58.7% coupling yield after purification. However, due to the low yield of its precursor 4, the chemically ligated ribozyme 9 was only obtained with a 8.1% overall yield to be compared with the 17.7% overall yield for RPI.3718 (Figure 11). CONCLUSION

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