WO2000018778A9 - Synthese d'acides nucleiques fondee sur une generation aleatoire de codons - Google Patents
Synthese d'acides nucleiques fondee sur une generation aleatoire de codonsInfo
- Publication number
- WO2000018778A9 WO2000018778A9 PCT/US1999/022436 US9922436W WO0018778A9 WO 2000018778 A9 WO2000018778 A9 WO 2000018778A9 US 9922436 W US9922436 W US 9922436W WO 0018778 A9 WO0018778 A9 WO 0018778A9
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- protecting group
- mononucleosides
- codons
- products
- protected
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- the invention relates to methods for chemically synthesizing DNA or RNA.
- Proteins with desired functions can be prepared using methods such as oligonucleotide-directed mutagenesis. Proteins with desired functions can also be selected from pools of randomly synthesized proteins, including proteins which are generated from random DNA template libraries.
- DNA libraries may also be generated using a variety of techniques. Such DNA libraries can be synthesized on a solid support (e.g., a CPG support), in a liquid phase, or in a combination solid-liquid phase (e.g., a PEG support). Most commonly, DNA libraries are prepared using a standard DNA synthesizer and a random mixture of all 4 nucleotides in each coupling step. By this approach, the trinucleotides, or codons, that correspond to the different amino acids, are randomly generated. This codon randomized DNA can then be transcribed into RNA, which is in turn used to synthesize polypeptides; the approach described above thus provides a means for generating a wide variety of DNA sequences and proteins products.
- a solid support e.g., a CPG support
- a combination solid-liquid phase e.g., a PEG support
- DNA libraries are prepared using a standard DNA synthesizer and a random mixture of all 4 nucleotides in each coupling step
- the invention features a method for generating a selected set of codons; the method includes the steps of: (a) providing a first set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where a subset A of the first set is protected with a protecting group A', and a subset B of the first set is protected with a protecting group B', where A' and B' are orthogonal protecting groups; (b) selectively removing protecting group A' from subset A; (c) coupling the products of step (b) with a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where the second set is protected with protecting group A'; (d) optionally removing protecting group A' from the products of step (c); (e) optionally coupling the products of step (d) with a third set of mononucleosides, where the third set is protected with protecting
- the selected set includes at least one codon corresponding to each of the 20 naturally-occurring amino acids; preferably, each of these codons corresponds to a highly expressed codon for one of the naturally-occurring amino acids.
- the selected set may also consist of trinucleotides coding only for a class of amino acids, e.g., hydrophobic amino acids, hydrophilic amino acids, basic amino acids, or acidic amino acids.
- the selected set may consist of trinucleotides coding for a mixture of amino acids, e.g., acidic and basic amino acids.
- steps (a) to (i) take place in the same reaction vessel; in addition, protecting groups A' and B' are two different groups and are preferably chosen from an acid-cleavable protecting group (for example a dimethoxytrityl group), a base-cleavable protecting group (for example, a fluorenylmethyloxycarbonyl group), or a fluoride-cleavable protecting group (for example, a silyl group).
- each of the codons terminates in a cytidine or a guanosine residue.
- the invention features a method for generating an oligonucleotide from a selected set of codons; the method includes the steps of: (a) providing a first set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where a subset A of the first set is protected with a protecting group A', and a subset B of the first set is protected with a protecting group B', where A' and B' are orthogonal protecting groups; (b) selectively removing protecting group A' from subset A; (c) coupling the products of step (b) with a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where the second set is protected with protecting group A'; (d) optionally removing protecting group A' from the products of step (c); (e) optionally coupling the products of step (d) with a third set of mononucleosides, where
- steps (a) to (k) take place in the same reaction vessel.
- the invention features a method for generating a selected set of codons including the steps of: (a) providing a first set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where a subset A of the first set is protected with a protecting group A', a subset B of the first set is protected with a protecting group B', and a subset C of the first set is protected with a protecting group C, where A', B', and C are orthogonal protecting groups; (b) selectively removing the protecting group A' from the subset A;(c) coupling the product formed in step (b) with a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where the second set is protected with the protecting group A'; (d) optionally removing the protecting group A' from the products of
- steps (a) to (o) take place in the same reaction vessel.
- one of the protecting groups A', B', and C is preferably an acid-cleavable protecting group (for example a dimethoxytrityl group)
- another of the protecting groups A', B', and C is preferably a base-cleavable protecting group (for example, a fluorenylmethyloxycarbonyl group)
- the last of the protecting groups A', B', and C is preferably a fluoride-cleavable protecting group (for example, a silyl group).
- the invention features a method for generating an oligonucleotide from a selected set of codons including the steps of: (a) providing a first set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where a subset A of the first set is protected with a protecting group A', a subset B of the first set is protected with a protecting group B', and a subset C of the first set is protected with a protecting group C, where A', B', and C are orthogonal protecting groups; (b) selectively removing the protecting group A' from the subset A; (c) coupling the product formed in step (b) with a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof, where the second set is protected with the protecting group A'; (d) optionally removing the protecting group A' from the products of step (c); (e) optionally coup
- steps (a) to (q) take place in the same reaction vessel.
- the invention features a method for generating, in the same reaction vessel, a selected set of codons; the method includes the steps of: (a) providing a first set of mononucleosides, mononucleotides, or dinucleotides, or mixture thereof; (b) adding a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof; (c) optionally adding a third set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof; and (d) optionally repeating step (c) to yield a selected set of codons.
- the selected set includes at least one codon having A or G at the third codon position; fewer than 3% of the codons in the selected set correspond to a stop codon.
- the selected set includes at least one codon for each of the 20 naturally-occurring amino acids; preferably, each codon corresponds to a highly-expressed codon for one of the naturally-occurring amino acids.
- the selected set may consist of one class of codons, e.g., hydrophobic amino acids.
- the selected set may consist of trinucleotides coding for a mixture of amino acids, e.g., acidic and basic amino acids.
- fewer than 2% of the codons correspond to a stop codon; more preferably, fewer than 1%, 0.5%, or O. /o, of the codons correspond to a stop codon.
- each of the codons terminates in a cytidine or a guanosine residue.
- any combination of couplings of mononucleosides, mononucleotides, and dinucleotides may be used to generated codons, which are trinucleotides.
- dinucleotides may be coupled with mononucleosides.
- Dinucleotides would not be coupled with dinucleotides, as that would generated tetranucleotides.
- the invention features a method for generating an oligonucleotide from a selected set of codons.
- the method includes the steps of: (a) providing a first set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof; (b) adding a second set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof; (c) optionally adding a third set of mononucleosides, mononucleotides, dinucleotides, or a mixture thereof; and (d) optionally repeating step (c) to yield a selected set of codons that includes at least one codon having A or G at the third codon position and in which fewer than 3% of the codons correspond to a stop codon.
- Steps (a), (b), (c), and (d) occur in the same reaction vessel; these steps are repeated until an oligonucleotide of the desired length is achieved.
- the selected set includes at least one codon for each of the 20 naturally-occurring amino acids, and fewer than 2% of the codons correspond to a stop codon.
- nucleoside is meant any sugar-base moiety, including sugar- base moieties in which one or more nitrogen atoms of the nitrogenous bases are protected, and/or in which the 5'-OH of the sugar is protected.
- Nucleosides also include nucleoside phosphoramidites and protected phosphoramidites.
- nucleotide is meant any sugar-phosphate-base moiety, as well as any derivatized sugar-phosphate-base moiety.
- One or more nitrogen atoms of the nitrogenous bases can be protected, and/or the 5'-OH of the sugar can be protected.
- Dinucleotides can include dinucleotide phosphoramidites; in addition, the internucleotide linkage may be protected.
- oligonucleotide is meant either a DNA sequence or an RNA sequence
- nucleic acid is meant either DNA or RNA.
- highly-expressed codons are meant the codons present in higher than normal abundance in highly expressed genes.
- stop codon is meant one of the DNA codons TAA, TGA, and TAG; and the RNA codons UAA, UGA, and UAG.
- a selected set of codons is meant a set of trinucleotide sequences where each trinucleotide has an assigned representation in the set.
- a selected set of codons may also be a set that is biased towards basic amino acids (e.g., His, Lys, Arg).
- the nucleosides and nucleotides used herein are often referred to with shorthand designations, in which the protecting group of the 5'-OH is superscripted.
- T C is used to represent ⁇ -benzoyl 5'-( -(4,4'- dimethoxytrityl) 2'-deoxycytidine or N'-benzoyl 5'-0-(4,4 , -dimethoxytrityl) 3'- O-(allyloxy diisopropylamino phosphinyl) 2'-deoxycytidine
- F G is used to represent N 2 -isobutyryl-5'-O-[(9-fluorenyl)methoxycarbonyl] 2'- deoxyguanosine or N 2 -isobutyryl-5'-O-[(9-fluorenyl)methoxycarbonyl] 3'-0- (allyloxy diisopropylamino phosphinyl) 2'-deoxyguanosine.
- the present invention provides a number of advantages over conventional techniques of nucleic acid synthesis.
- the methods described herein provide control over codon format (trinucleotide sequences) as well as control over the representation of the codon in a selected set.
- the invention can therefore generate sets of codons that contain at least one codon for each of the naturally-occurring amino acids and, importantly, can generate libraries that are substantially free of stop codons.
- the invention also provides DNA consisting essentially of highly-expressed codons and, as noted above, free of stop codons, which can be efficiently translated to polypeptides of desired lengths.
- control over codon representation allows for the synthesis of DNA templates that can be used to generate proteins rich in selected amino acids, for example, hydrophobic amino acids, which can be instrumental in protein design techniques.
- FIGURES 1, 2, 3, 4, 5, 6, 7, 8, and 9 are each illustrations of coupling sequences for the synthesis of codon libraries.
- sequence of bases in DNA and its RNA counterpart determines the sequence of the amino acids in the protein synthesized from this DNA. Sequences of three bases, referred to as codons, correspond to different amino acids. During translation, these codons are read from the 5' end to the 3' end; the resulting protein has an amino acid sequence that corresponds to the sequence of codons.
- TAA, TAG, and TGA which correspond to the RNA codons UAA, UAG, and UGA
- these codons signal release factors to terminate protein synthesis.
- the presence of stop codons therefore leads to termination of protein synthesis before the entire DNA sequence is translated.
- the invention features convenient methods for the controlled synthesis of codon randomized nucleic acids, such as DNA, in which the presence of stop codons can be avoided.
- the DNA strand can be used as a template for the synthesis of a complementary DNA strand, which in turn can serve as a template for the synthesis of the corresponding messenger RNA.
- messenger RNA can be synthesized directly using the methods of the invention.
- a desired set of codons, as well as the desired frequency of each codon in the set is first chosen.
- the set can include, for example, the most highly-expressed codons for each of the 20 naturally-occurring amino acids, in equal distribution.
- Highly expressed DNA codons in eukaryotic translation systems typically exhibit either 2'-deoxycytidine (C) or 2'-deoxyguanosine (G) at the 3' end (that is, at the third codon position).
- Another desired set can include, for example, at least one codon for each of the 20 naturally-occurring amino acids, and in which hydrophobic amino acids are twice as abundant.
- a selected set of highly expressed codons for all 20 naturally-occurring amino acids can be prepared in which all of the codons have a C or G at the third position. Two of the three stop codons, TAA and TGA, are therefore readily excluded from this set.
- stop codons has a C at the third position; codons ending in C can therefore be randomly generated without the introduction of stop codons.
- the generation of a set of codons ending in C can produce codons for fifteen of the naturally-occurring amino acids.
- the present invention also features methods for generating libraries of codons by using nucleosides and nucleotides with different 5'-protecting groups as building blocks.
- the different 5'-protecting groups can be cleaved under orthogonal conditions.
- the conditions for cleaving one 5'-protecting group do not cleave the other 5'- protecting groups.
- An example of one pair of orthogonal protecting groups includes a dimethoxytrityl group (DMT or T), which is cleaved under acidic conditions, and a fluorenylmethyloxycarbonyl group (Fmoc or F), which is cleaved under basic conditions.
- a set of orthogonal protecting groups is the set including a dimethoxytrityl group (DMT or T), which is cleaved under acidic conditions, a fluorenylmethyloxycarbonyl group (Fmoc or F), which is cleaved under basic conditions, and a silyl group (S), which is cleaved with fluoride.
- DMT or T dimethoxytrityl group
- Fmoc or F fluorenylmethyloxycarbonyl group
- S silyl group
- a mixture of nucleosides is treated with acid.
- the DMT-protected nucleosides are deprotected, while the Fmoc protected nucleosides remain protected.
- nucleotides are added to this mixture, they couple only with the deprotected nucleosides, allowing for coupling specificity.
- a mixture of 7v rf -benzoyl-5 , -0-(4,4'-dimethoxytrityl)- 2'-deoxycytidine ( T C) and N 2 -isobutyryl-5'-O-[(9-fluorenyl)methoxycarbonyl]- 2'-deoxyguanosine ( F G) is treated with acid.
- the DMT group is cleaved from the C mononucleosides, thus leaving them free to couple with nucleotides.
- the Fmoc of the G mononucleosides remains attached. Since none of the stop codons end in C, trinucleotides may be randomly generated at this step without the introduction of stop codons.
- nucleoside phosphoramidites are used for the coupling reactions.
- the internucleotide linkages can be protected with a protecting group, such as an allyl moiety.
- the allyl protecting group is stable toward both acid and base, but can be cleaved with aqueous ammonia or by palladium (Pd(0)) catalysis.
- Example 1 A Synthesis of ⁇ benzoyl-S'-O- ⁇ '-riimethoxytritylY 3'- O-(a11yloxy diisopropylamino phosphinyl) 2'-deoxycytidine ( T C)
- Example 1 B Alternative synthesis of DMT-allyl dC phosphoramidite monomer
- the reaction mixture was then extracted with 5% NaHC0 3 (3 x 50 mL), H 2 0 (2 x 50 mL), and dried with Na j SO ⁇
- the Na 2 S0 4 was removed by filtration, and the organics were concentrated to 50 mL under reduced pressure before loading onto a silica gel column (200 g).
- the column was eluted with EtOAc/heptane/TEA (49/50/1 v:v).
- Fractions containing the desired product were combined and evaporated under reduced pressure to yield an oily residue, which was applied to another silica gel column (200 g) and eluted with a stepwise gradient of EtOAc (25 ⁇ 75%) in heptane containing 1% TEA.
- the chemical stability of the new DMT-allyl dC phosphoramidite monomer was monitored by preparing a 0.1M solution in CDC1 3 and collecting the 31 P NMR spectrum at 24 hour intervals. The monomer was determined to be stable for at least 8 days (i.e., no change in spectrum between 300 and -50 ppm).
- the coupling ability of the new monomer was evaluated by solid-phase synthesis of the sequence 5'-d(C 9 T) on an automated DNA synthesizer (Expedite 8909, PerSeptive Biosystems) using a standard coupling protocol provided by the manufacturer, except that the monomer coupling time was increased to 120 seconds.
- the solid support from the two syntheses was divided into five portions and treated with 1.5 mL of one of the following at the indicated temperature: concentrated ammonium hydroxide at room temperature, concentrated ammonium hydroxide at 55 °C, a mixture of concentrated ammonium hydroxide in ethanol (3:1 v/v) at 55° C, a mixture of t-butyl amine/methanol/water (1:1:2 v/v) at 55 °C, 2M anhydrous ammonia in methanol at 55 °C.
- Example 2 A Synthesis of N -isobutyry1-5'-( -[(9- fluorenyl)methoxycarbonyl] 3'-Q-fallyloxy diisopropylamino phosphinyl) 2'-deoxyguanosine ( F G)
- the product is then converted to the phosphoramidite (also referred to as F G) using the reaction conditions described in Lehmann et al., Nucleic Acids Res., Vol. 17, No. 7, 2379-2390 (1989).
- N 6 -benzoyl-5'-0-[(9-fluorenyl)methoxycarbonyl] 3'-0-(allyloxy diisopropylamino phosphinyl) 2'-deoxyadenine ( F A) is prepared using the same reaction conditions.
- Example 2B Alternative synthesis of Fmoc-allyl dG phosphoramidite monomer and its application in D ⁇ A synthesis
- the chemical stability of the new Fmoc-allyl dG phosphoramidite monomer was monitored by preparing a 0.1M solution in CDC1 3 and collecting the 31 P NMR spectrum at 24 hour intervals. The monomer was 10% degraded after 1 day and 50% degraded after 3 days, as indicated by the appearance and growth of a new peak at 153 ppm resulting from spontaneous loss of the 5'-Fmoc group. As most syntheses are completed within several hours, the stability of the Fmoc-allyl dG phosphoramidite was deemed suitable.
- the coupling ability of the new monomer was evaluated by solid-phase synthesis of the sequence 5'-d(G 9 T) on an automated DNA synthesizer (Expedite 8909, PerSeptive Biosystems).
- the standard synthesis protocol provided by the manufacturer was modified to increase the coupling time (900 sec), increase the capping step (120 sec), increase the oxidation time (60 sec), and deliver the 5'-Fmoc deprotection reagent for 120 seconds from an auxiliary bottle position.
- Both 0.1M DBU in acetonitrile and O.lM piperidine in anhydrous DMF were evaluated as 5'-Fmoc deprotection reagents.
- the completed 5'-d(G 9 T) sequences were deprotected in concentrated ammonium hydroxide for 18 hours at 55 °C, concentrated in a Speed- Vac, analyzed by anion-exchange HPLC under denaturing conditions (Dionex DNAPac PA- 100 column, sodium chloride gradient in 25 mM NaOH, pH 12.4), and compared to a control sequence synthesized with standard DMT-dG cyanoethyl phosphoramidites.
- 0.1M Piperidine in DMF was the preferred 5'-Fmoc deprotection reagent.
- Example 3A Synthesis of N -isobutyry1-5'-O-[rrimethylsi1y1] 3'- ⁇ - (allyloxy diisopropylamino phosphinyl) 2'-deoxyg ⁇ nosine ( S G)
- the product is then converted to the phosphoramidite (also referred to as S G) using the reaction conditions described in Example 1.
- Example 3B Synthesis of silyl-allyl dG phosphoramidite monomer and its application in DNA synthesis
- N 2 -Isobutyryl-2'-deoxyguanosine (6.75 g, 20 mmol) was evaporated from pyridine (3 x 100 mL), dissolved in anhydrous pyridine (75 mL) and cooled to 0°C.
- Bis(trimethylsiloxy)cyclododecyloxy-silyl chloride (8.5 g, 22 mmol) was added to the stirred solution. After two hours the reaction mixture was concentrated to dryness under reduced pressure and resuspended in CH 2 C1 2 (100 mL).
- the coupling ability of the new Silyl-allyl dG monomer was evaluated by solid-phase synthesis of the sequence 5'-d(G 9 T) on an automated DNA synthesizer (Expedite 8909, PerSeptive Biosystems) using a polystyrene solid support (PE BioSystems, Foster City, CA).
- the standard 0.2 ⁇ mole cyanoethyl phosphoramidite synthesis protocol provided by the manufacturer was modified to accommodate the new chemistries.
- the modified protocol contained longer monomer coupling steps (240 sec), longer wash times (120 sec), and new cycles to deliver the non-standard Silyl deprotection reagent (HF/TEA, 1.1M:1.6M in DMF) from an auxiliary bottle position.
- the oil is co- evaporated from toluene (twice), ethanol, then chloroform, and subjected to a short column chromatography (silica gel) eluting with a gradient of 0-5% methanol in chloroform. Fractions containing the major product are collected, concentrated to a foam, dissolved in chloroform, precipitated with pentane, filtered, and then dried under vacuum.
- the DMT protecting group is then cleaved as follows.
- the product is dissolved in 75 ml CH 2 Cl 2 /MeOH (8:1 v/v); Amberlyst® 15 ion exchange resin is then added in portions until the surface of the resin remains orange colored.
- the suspension is stirred 24 hours, the resin is filtered off, and the solution is concentrated in vacuo.
- the product is precipitated twice from petroleum ether (500 ml) at 40-60°C.
- ⁇ -benzoyl-3'-O-tert-butyl-dimethylsiryl 2'-deoxyadenine N 2 - isobutyryl-3'-0-tert-butyl-dimethylsilyl 2'-deoxyguanosine, and ⁇ -benzoyl-3'- O-tert-butyl-dimethylsilyl 2'-deoxycytidine are prepared using the same reaction conditions.
- a solution containing a mixture of 15 mmol 3'-0-tert-butyl- dimethylsilyl 2'-deoxythymidine and 24 mmol tetrazole is dried by repeated coevaporation with acetonitrile/toluene. The mixture is then dissolved in 50 ml dry acetonitrile. 15 mmol N 6 -benzoyl-5'-0-(4,4 , -dimethoxytrityl) 3'-0- (allyloxy diisopropylamino phosphinyl) 2'-deoxyadenine, which is pre-dried by repeated coevaporation with toluene, in 30 ml dry acetonitrile is added.
- reaction is followed by TLC. If the reaction does not go to completion, additional phosphoramidite can be added.
- the TBDMS ether protecting group is cleaved as follows.
- the product is dissolved in 40 ml THF. 30 mmol tetrabutylammonium fluoride is added, and the reaction mixture is stirred 1 hour at 25 °C.
- the THF is evaporated in vacuo; water is then added to the concentrated reaction mixture.
- the resulting mixture is extracted with CH 2 C1 2 (3 l00 ml).
- the combined organic layers are dried over ⁇ a 2 S0 4 , filtered, and concentrated.
- the product is then purified with column chromatography (silica gel, using methanol in
- Example 6 Synthesis of ⁇ -benzoyl-5'-O-r4.4'-dime,thoxyrrity1)-2'- deoxycytidine 3'-O-succinic acid N'-benzoyl-5 l -O-(4,4 , -dimethoxytrityl)-2'-deoxycytidine and succinic anhydride (10-fold excess) are dissolved in DMF and stirred at 70 °C for 40 hours. The reaction is monitored by TLC (silica gel, development in ether, then chloroform methanol 9:1). After completion of the reaction, the reaction mixture is taken up in methylene chloride, then washed with 20% aqueous citric acid solution.
- TLC sica gel, development in ether, then chloroform methanol 9:1
- Example 7 Functionalization of support A glass support for use in D ⁇ A synthesis is treated with Fmoc- sarcosine in the presence of dicyclohexylcarbodiimide, followed by removed of the Fmoc group with piperidine/DMF.
- the support is separated by filtration, washed with methylene chloride and diethyl ether. Unreacted amino groups are capped by treatment of the support with a mixture of THF/lutidine/acetic anhydride (8:1 :1) and N-methylimidazole in THF. The support is then washed with methylene chloride and diethyl ether, and dried in vacuo.
- the codons are built up from the 3'-end, as shown in Figure 1, using solid phase synthesis.
- a solid phase synthesizer is used, according to the manufacturer's instructions.
- a 16:5 mixture of ⁇ -benzoyl-S'-O- ⁇ '-dimethoxytrityl) ⁇ '- deoxycytidine ( T C) and N 2 -isobutyryl-5'-O-[(9-fluorenyl)methoxycarbonyl]-2'- deoxyguanosine ( F G) is attached to a support, as described in Examples 6 and 7.
- trichloroacetic acid is added to cleave the trityl protecting groups from the T C mononucleosides. Since the Fmoc protecting group is not labile under acidic conditions, the F G mononucleosides remain protected, and therefore unreactive.
- the trityl protecting groups of the dinucleotides are then cleaved with acid.
- the dinucleotides are coupled with a 1:1:1:1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the result is a mixture of 16 unique codons, each corresponding to a different amino acid (with the exception of TTC and AGC, which both represent serine), and F G mononucleosides.
- the Fmoc protecting groups of the G mononucleosides are then cleaved with l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), as described in Lehmann et al., Nucleic Acids Res. 17:2379 (1989).
- DBU l,8-diazabicyclo[5.4.0]undec-7-ene
- the trityl protecting groups of the trinucleotides are not labile under basic conditions; the trinucleotides therefore remain unreactive.
- the deprotected G mononucleosides are coupled with a 3 : 1 : 1 mixture of F A mononucleoside phosphoramidites, T TG dinucleotide phosphoramidite, and T AT dinucleotide phosphoramidite, and the products of the coupling reactions are oxidized.
- the result is two more trinucleotide codons, and F AG dinucleotides.
- the Fmoc protecting groups of the dinucleotides are once again cleaved with base, the dinucleotides are coupled with a 1 :1 : 1 mixture of T A, 1 C, and T G mononucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the nucleotide T is omitted, as the inclusion of this nucleotide at this point would result in the synthesis of a TAG codon.
- the end result of the successive deprotection and coupling reactions is a mixture of 21 codons, each corresponding to one of the 20 naturally occurring amino acids. All 20 amino acids are represented, and only one amino acid is represented twice.
- the invention therefore provides a synthesis of a codon set in which all of the amino acids are represented approximately equally. Most importantly, the set contains substantially no stop codons.
- the trityl group can be removed from the trinucleotides, and a mixture of T C and F G nucleoside phosphoramidites can be added. The process for synthesizing the codons can then be repeated until DNA of the desired length is achieved.
- the standard 0.2 ⁇ mole cyanoethyl phosphoramidite synthesis protocol provided by the manufacturer was modified to accommodate the new chemistries.
- the modified protocol contained longer monomer coupling steps (240 sec), longer wash times (120 sec), and new cycles to deliver the non-standard Fmoc deprotection reagent (0.1M piperidine in anhydrous DMF).
- the 5'-DMT was removed with 3% dichloroacetic acid in CH 2 C1 2 and DMT-C monomer was then delivered to the column to extend the CCT sequence to CCCT.
- a 0.1M piperidine solution in DMF was delivered to the column to remove the 5'-Fmoc protecting group.
- DMT-C monomer was again added to the column to form CGCT from the remaining GCT sequence.
- the terminal 5'-DMT was removed with 3% dichloroacetic acid in CH 2 C1 2 and the CPG support was treated with concentrated ammonium hydroxide at 55° C for 16 hours. The solution was finally cooled, concentrated to dryness on a Speed- Vac, and taken up in water.
- the tetramer standard (prepared with conventional cyanoethyl phosphoramidites and the manufacturer's S coupling cycle at base position three) produced 2.4:0.6: 1.0 for the normalized ratio of C:G:T nucleosides, respectively, compared to a theoretical value of 2.5:0.5: 1.
- the same tetramer prepared via the acid/base orthogonal deprotection scheme produced 2.2:0.8:1.0 for the normalized ratio of C:G:T nucleosides.
- the 15-mer 5'-d(ACGTGGCTGAACSCT), where S is either G or C, was also synthesized on an automated DNA synthesizer using the same acid/base orthogonal deprotection scheme described above.
- the terminal 5'-DMT was removed with 3% dichloroacetic acid in CH 2 C1 2 , and the CPG support was treated with concentrated ammonium hydroxide at 55° C for 16 hours. The solution was finally cooled, concentrated to dryness on a Speed- Vac, and taken up in water.
- the ratio of the peak areas was 0.61 :0.39
- the ratio of the peak areas was 0.51 :0.49.
- Example 8C Synthesis of CCCJ GC codons (Pro/Arg) via acid/fluoride orthogonal deprotection
- the modified protocol contained longer monomer coupling steps (240 sec), longer wash times (120 sec), and new cycles to deliver the non-standard silyl deprotection reagent (HF/TEA, 1. IM: 1.6M in DMF) over 180 seconds.
- the 5'-DMT was removed with 3% dichloroacetic acid in CH 2 C1 2 and DMT-C monomer was delivered to the column to extend the CCT sequence to CCCT.
- an HF/TEA mixture in DMF (1.1M:1.6M) was delivered to the column to remove the 5'-Silyl protecting group.
- DMT-C monomer was again added to the column to form CGCT from the remaining GCT sequence.
- the terminal 5'-DMT was removed with 3% dichloroacetic acid in CH 2 C1 2 and the CPG support was treated with concentrated ammonium hydroxide at 55 °C for eight hours. The solution was finally cooled and concentrated on a Speed- Vac.
- the tetramer standard (prepared with conventional cyanoethyl phosphoramidites and the manufacturer's S coupling cycle at base position three) produced 2.0: 1.0: 1.0 for the normalized ratio of C:G:T nucleosides, respectively, compared to a theoretical value of 2.5:0.5:1.
- the same tetramer prepared via the acid/base orthogonal deprotection scheme produced the normalized ratio 1.9: 1.1 : 1.0 for C:G:T nucleosides.
- the ratio of the peak areas at 260 nm was 0.34:0.66, whereas in the case of the standard (prepared with conventional cyanoethyl phosphoramidites and the manufacturer's S coupling cycle at base position three) the ratio of the peak areas was 0.4:0.6.
- Example 9 Removal of oligonucleotide from support
- the support is treated with concentrated ammonia at 70 °C for 2 hours in a tightly closed Eppendorf tube, to cleave the oligonucleotides from the support.
- the ammonia solution is evaporated on a speed- vac concentrator.
- the residue is taken up in water and centrifuged (15 minutes, 0°C). DNA is precipitated from the supernatant by the addition of dioxane and THF. After centrifuging (15 minutes, 0°C), the pellet is dissolved in water.
- the product DNA is purified by reverse-phase HPLC.
- the support material is treated under argon with
- examples 10-13 are carried out using the general methods described in Example 8; the successive coupling reactions take place in the same reaction vessel.
- a 14:6 mixture of T C and F G is attached to a support, as described in Examples 6 and 7.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are then coupled with a 1 : 1 :1:1:1:1:1: 1 :1 :1:1:1 mixture of T AA, T CA, T GA, T TA, T AC, T CC, T GC, T AG, T GG, T TG, T AT, T CT, T GT, and T TT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the result of these reactions is a mixture of 14 unique codons, each representing a different amino acid, and F G mononucleoside, as shown in Figure 2.
- the Fmoc protecting groups of the G mononucleosides are then cleaved with DBU, as described in Example 6.
- the deprotected mononucleosides are coupled with a 1 :1 : 1:1 :1:1 mixture of T AA, T CA, T GA, T AG, T TG, and T AT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the end result of these coupling reactions is a mixture of 20 unique trinucleotides, each representing a codon for one of the 20 naturally-occurring amino acids, as shown in Figure 2. Once again, no stop codons are present in the mixture.
- This process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- a 16:5 mixture of T C and F G mononucleosides is attached to a support.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are then coupled with a 1 : 1 : 1 :1 mixture of
- T A, T C, T G, and T T nucleoside phosphoramidites and the products of the coupling reactions are oxidized.
- the result of these reactions is a 1 : 1 : 1 : 1 mixture of T AC, T CC, T GC, and T TC dinucleotides, and F G mononucleoside.
- the trityl protecting groups of the dinucleotides are then cleaved with acid.
- the dinucleotides are coupled with a 1 : 1 : 1 : 1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the result is a mixture of 16 unique codons, each representing a different amino acid (with the exception of TTC and AGC, which both correspond to serine), and F G mononucleoside.
- the protecting groups of the F G mononucleosides are then cleaved with DBU.
- the G mononucleosides are coupled with a 1 : 1 : 1 : 1 : 1 mixture of T AA, T CA, T GA, T TG, and T AT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the end result of the successive deprotection and coupling reactions is a mixture of 21 codons.
- the process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- a 16:6 mixture of T C and F G mononucleosides is attached to a support, and the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are coupled with a 1 : 1 : 1 : 1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the result of these reactions is a 1:1:1:1 mixture of T AC, T CC, T GC, and T TC dinucleotides, and F G mononucleoside.
- the trityl protecting groups of the dinucleotides are then cleaved with acid.
- the dinucleotides are coupled with a 1 : 1 :1 : 1 mixture of T A, T C, T G, and T T mononucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the protecting groups of the F G mononucleosides are then cleaved with DBU.
- the G mononucleosides are coupled with a 3 : 1 : 1 : 1 mixture of F A nucleoside phosphoramidite and T TG, T AT, and T CU dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the Fmoc protecting groups are cleaved from the dinucleotides, and a 1 :1 :1 mixture of T A, T C, and T G mononucleoside phosphoramidites is added; the products of the coupling reactions are then oxidized.
- the result of these successive deprotection and coupling reactions is a mixture of 22 codons, each corresponding to an amino acid.
- the synthetic scheme results in the generation of a set of codons in which the amino acids Ser and Leu are twice as abundant as the other naturally occurring amino acids. This distribution is close to the amino acid distribution typically found in biological proteins.
- the process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- Example 13 Synthesis of trinucleotides
- a 16:6 mixture of T C and F G mononucleotides is attached to a support.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are coupled with a 1 :1 :1 :1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the result of these reactions is a 1:1:1:1 mixture of T AC, T CC, T GC, and T TC dinucleotides, and F G mononucleoside.
- the trityl protecting groups of the dinucleotides are then cleaved with acid.
- the dinucleotides are coupled with a 1 : 1 : 1 : 1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the protecting groups of the F G mononucleotides are then cleaved with DBU.
- the G mononucleosides are coupled with a 1 :1 :1 : 1 :1 :1 mixture of T AA, T CA, T GA, T TG, T AT, and T CU dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the result of the successive deprotection and coupling reactions is a mixture of 22 codons.
- the synthetic scheme results in the generation of a set of codons in which the amino acids Ser and Leu are twice as abundant as the other naturally occurring amino acids. This distribution represents the amino acid distribution found in biological proteins.
- the process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- a 16:3:2 mixture of T C, F G, and S G mononucleosides is attached to a support.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are coupled with a 1 : 1 : 1 : 1 mixture of T A,
- T C, T G, and T T nucleoside phosphoramidites and the products of the coupling reactions are oxidized.
- the result of these reactions is a 1 : 1 : 1 : 1 mixture of T AC, T CC, T GC, and T TC dinucleotides, F G mononucleosides, and S G mononucleosides .
- the trityl protecting groups of the dinucleotides are cleaved with acid.
- the dinucleotides are coupled with a 1:1 :1:1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the protecting groups of the F G mononucleotides are cleaved with DBU.
- the G mononucleosides are coupled with F A mononucleoside phosphoramidite, and the products of the coupling reactions are oxidized.
- the Fmoc protecting groups are again cleaved.
- the dinucleotides are coupled with a 1: 1:1 mixture of T A, T C, and T G mononucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the silyl protecting groups are cleaved with anhydrous tetra-n- butylammonium fluoride.
- the G mononucleosides are coupled with a 1:1 mixture of F G and S T mononucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the Fmoc protecting group of the dinucleotide is cleaved, and the dinucleotide is coupled with T T mononucleoside phosphoramidite. The product of the coupling reaction is oxidized. Finally, the silyl group of the S TG dinucleotide is cleaved. The dinucleotide is coupled with T A, and the product is oxidized.
- the result of the successive deprotection and coupling reactions is a mixture of 21 codons.
- the process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- a 6: 1 mixture of T C and F G mononucleosides is attached to a support, and the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are coupled with a 1 : 1 : 1 : 1 : 1 : 1 mixture of T CC, T GC, T AT, T CT, T GT, and T TT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the Fmoc protecting group of the F G mononucleoside is then cleaved.
- the mononucleoside is coupled with a T AT dinucleotide phosphoramidite, and the product of the coupling reaction is oxidized.
- a 16:5 mixture of T C and F G mononucleosides is attached to a support.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are then coupled with a 1 :1 : 1 :2 mixture of
- T A, T C, T G, and T T nucleoside phosphoramidites and the products of the coupling reactions are oxidized.
- the result of these reactions is a 1 :1:1:2 mixture of T AC, T CC, T GC, and T TC dinucleotides, and F G mononucleoside.
- the trityl protecting groups of the dinucleotides are then cleaved with acid.
- the dinucleotides are coupled with a 1 :1:1 : 1 mixture of T A, T C, T G, and T T nucleoside phosphoramidites, and the products of the coupling reactions are oxidized.
- the protecting groups of the F G mononucleosides are then cleaved with DBU.
- the G mononucleosides are coupled with a 1 : 1 : 1 : 1 : 1 mixture of
- T AA, T CA, T GA, T TG, and T AT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the end result of the successive deprotection and coupling reactions is a mixture of 20 codons; the codons ATC, CTG, GTC, and TTC, which correspond to the hydrophobic amino acids He, Leu, Val, and
- Example 17 Synthesis of codons with a bias for basic amino acids
- a 14:6 mixture of T C and F G mononucleosides is attached to a support.
- the trityl protecting groups are cleaved with trichloroacetic acid.
- the C mononucleosides are then coupled with a 1:2:1:1:1:1: 1 :1 :1 : 1 : 1 : 1 : 1 mixture of T AA, T CA, T GA, T TA, T AC, T CC, T GC, T AG, T GG, T TG, T AT, T CT, T GT, and T TT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the protecting groups of the F G mononucleosides are then cleaved with DBU.
- the G mononucleosides are coupled with a 2: 1 :1:2: 1 : 1 mixture of T AA, T CA, T GA, T AG, T TG, and T AT dinucleotide phosphoramidites, and the products of the coupling reactions are oxidized.
- the end result of the successive deprotection and coupling reactions is a mixture of 20 codons; the codons CAC, AAG, and AGG, which correspond to the basic amino acids His, Lys, and Arg, are represented twice.
- the process for synthesizing the codons can be repeated until DNA of the desired length is achieved.
- the resulting DNA will code for proteins with a high percentage of basic amino acids.
- Example 10 For example, after a group of codons is prepared as described in Example 8, the scheme described in Example 10 may be used to generate the next set of codons. This process may be continued until DNA of the desired length is achieved.
- the trinucleotides generated by any approach may be cleaved from the support using concentrated ammonia at room temperature.
- the 3'-OH group is then derivatized with allyloxy bis- (diisopropylamino)phosphine to yield the trinucleotide phosphoramidite, and the trinucleotide phosphoramidites are then used as building blocks to synthesize DNA.
- the methods of the invention may be used for any application in which nucleic acid synthesis is required.
- these methods can be used in the synthesis of single-stranded DNA.
- this DNA serve as a template for the synthesis of a complementary DNA strand, which can in turn serves as a template for messenger RNA synthesis.
- the methods of the invention find use, for example, in techniques of randomized cassette mutagenesis of proteins, phage display techniques, ribosome display techniques, and protein-nucleic acid fusion techniques.
- Codon-randomized DNA can also be used in cellular cultures (in vivo) for protein expression, or for in vitro applications using, for example, T7 RNA polymerase, and in vitro translation systems.
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Abstract
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AU62704/99A AU6270499A (en) | 1998-09-29 | 1999-09-28 | Synthesis of codon randomized nucleic acids |
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AU1449499A (en) | 1997-10-31 | 1999-05-24 | Maxygen, Incorporated | Modification of virus tropism and host range by viral genome shuffling |
US6222030B1 (en) * | 1998-08-03 | 2001-04-24 | Agilent Technologies, Inc. | Solid phase synthesis of oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection |
EP1176151B1 (fr) | 2000-07-28 | 2014-08-20 | Agilent Technologies, Inc. | Synthèse des polynucléotides utilisant la chimie combinée d'oxidation et de protection |
WO2002010201A2 (fr) | 2000-07-31 | 2002-02-07 | Active Motif | Administration de molecules dans des cellules par mediation peptidique |
US6689568B2 (en) | 2001-02-01 | 2004-02-10 | Agilent Technologies, Inc. | Capture arrays using polypeptide capture agents |
US6852850B2 (en) | 2001-10-31 | 2005-02-08 | Agilent Technologies, Inc. | Use of ionic liquids for fabrication of polynucleotide arrays |
US6858720B2 (en) | 2001-10-31 | 2005-02-22 | Agilent Technologies, Inc. | Method of synthesizing polynucleotides using ionic liquids |
EP1860115A4 (fr) * | 2005-02-28 | 2010-09-22 | Tokyo Inst Tech | Derive oligonucleotidique, sonde de detection du gene, et puce a adn |
WO2016077123A1 (fr) | 2014-11-10 | 2016-05-19 | Moderna Therapeutics, Inc. | Optimisation d'acides nucléiques à plusieurs paramètres |
US20210206818A1 (en) | 2016-01-22 | 2021-07-08 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
EP3896164A1 (fr) | 2016-05-18 | 2021-10-20 | ModernaTX, Inc. | Polynucléotides codant pour l'alpha-galactosidase a pour le traitement de la maladie de fabry |
US20190298657A1 (en) | 2016-05-18 | 2019-10-03 | Modernatx, Inc. | Polynucleotides Encoding Acyl-CoA Dehydrogenase, Very Long-Chain for the Treatment of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency |
MA45052A (fr) | 2016-05-18 | 2019-03-27 | Modernatx Inc | Polynucléotides codant pour jagged1 pour le traitement du syndrome d'alagille |
ES2932516T3 (es) | 2016-05-18 | 2023-01-20 | Modernatx Inc | Combinaciones de ARNm que codifican polipéptidos inmunomoduladores y usos de los mismos |
WO2017201349A1 (fr) | 2016-05-18 | 2017-11-23 | Modernatx, Inc. | Polynucléotides codant pour la citrine pour le traitement de la citrullinémie de type 2 |
RS63912B1 (sr) | 2016-05-18 | 2023-02-28 | Modernatx Inc | Polinukleotidi koji kodiraju interleukin-12 (il12) i njihove upotrebe |
EP3458104A1 (fr) | 2016-05-18 | 2019-03-27 | Modernatx, Inc. | Polynucléotides codant pour la porphobilinogène désaminase destinés au traitement de la porphyrie intermittente aiguë |
US11001861B2 (en) | 2016-05-18 | 2021-05-11 | Modernatx, Inc. | Polynucleotides encoding galactose-1-phosphate uridylyltransferase for the treatment of galactosemia type 1 |
CA3063723A1 (fr) | 2017-05-18 | 2018-11-22 | Modernatx, Inc. | Polynucleotides codant pour des polypeptides d'interleukine-12 (il12) ancres et leurs utilisations |
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US5430138A (en) * | 1990-07-27 | 1995-07-04 | Chiron Corporation | Hydroxyl-protecting groups attached to cytidine nucleotide compounds which are orthogonally removable |
US5889136A (en) * | 1995-06-09 | 1999-03-30 | The Regents Of The University Of Colorado | Orthoester protecting groups in RNA synthesis |
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