US005436143A United States Patent 19) 11 Patent Number: 5,436,143 Hyman - 45 Date of Patent: Jul. 25, 1995 54 METHOD FORENZYMATICSYNTHESIS OF cleotides', in Oligonucleotide Synthesis: A Practical Ap OLGONUCLEOTIOES proach, M. J. Gait ed., pp. 185-197 (1984). Mudrakovskaya et al., “RNA Ligase of Bacteriophage 76 Inventor: Edward D. Hyman, 2100 Sawmill T4. VII: A solid pahse enymatic synthesis of Rd., Apt. 4-103, River Ridge, La. oligoribonucelotides', Biorg. Khim., 17: 819-822 701.23 (1991). (21) Appl. No.: 995,791 Stuart et al., “Synthesis and Properties of Oligodeox ynucleotides with an AP site at a preselected location', 22 Filed: Dec. 23, 1992 Nulceic Acids Res. 15: 7451-7462 (1987). 51) Int. Cl. .............................................. C12P 19/34 Norton et al., "A ribonuclease specific for 2'-O-Me 52 U.S. C. .................................. 435/91.2; 435/91.1; thyltaed Ribonulceic Acid', J. Biol. 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(56) References Cited Ohtsuka et al., “A new method for 3'-labelling of U.S. PATENT DOCUMENTS polyribonucelotides by phosphorylation with RNA 3,850,749 11/1974 Kaufmann et al. ligase and its application to the 3'-modification for join 4,661,450 4/1987 Kempe et al. .. ... 435/1723 ing reactions', Nulceic Acids Res. 6: 443-454 (1979). 4,987,071 1/1991 Cech et al. ............................ 435/91 (List continued on next page.) OTHER PUBLICATIONS Primary Examiner-Margaret Parr Uhlenbeck et al. (1982) The Enzymes, vol. XV, pp. Assistant Examiner-Ardin H. Marschel 31-58. Attorney, Agent, or Firm-Oppendahl & Larson Hoffmann et al. (1987) Nucleic Acids Research, vol. 15, 57 ABSTRACT No. 3, pp. 5289-5303. Enzymatic synthesis of oligonucleotides may be per Shum et al., "Simplified method for large scale enzy formed in a single vessel without intermediate purifica matic synthesis of oligoribonucleotides', Nucleic Acids tion, by the steps of: Res. 5: 2297-2311 (1978). (a) combining a nucleotide primer sequence and a Schott et al., “Single-step elogation of oligodeoxynu blocked nucleotide in the presence of a chain ex cleotides using terminal deoxynucleotidyl transferase', tending enzyme whereby a reaction mixture is Eur. J. Biochem. 143: 613-620 (1984). formed containing the blocked nucleotide coupled Mackey et al., “New approach to the synthesis of to the nucleotide primer sequence at its 3' end; polyribonucleotides of defined sequence', Nature 233: (b) inactivating the chain extending enzyme; 551-553 (1971). (c) removing the blocking group from the primer Hinton et al., “The preparative synthesis of oligodeox blocked nucleotide to form a primer-nucleotide y-ribonucleotides using RNA ligase', Nucleic Acids product; and converting any unreacted blocked Res. 10: 1877-1894 (1982). nucleotide to an unreactive form which is substan England et al., "Dinucleotide pyrophosphates are sub tially less active as a substrate for the chain extend strates for T4-induced RNA ligase', Proc. Nat'l Acad ing enzyme than the blocked nucleotide. Sci. (USA) 74: 4839-4842 (1977). Beckett et al., "Enzymatic Synthesis of Oligoribonu 18 Claims, 7 Drawing Sheets UNCONTROLLED METHOO prince - ruckootico Tronsorse enrymercuteer primof + (prime?-- 1) + (odine?--2} + per+3+...ole. Purity permet+h) production side products petre+1) Rupect crickeunincorrucleodernihess compolo BLOCKED METHOD portex 4 blocked-nuclectice Tronaerose enzymercubeson part of + (pritof+theocod-blocked-nucleode rocMctorytowdolonerose Remoral of Blocking group chemicolorenzymolic) pairnor 4 prTed) + nucsorse l Purtynor) product beinricococcipatna prime 4-1 Rupood cyclotundocreocco sness corre 5,436,143 Page 2 OTHER PUBLICATIONS thesis with T4 RNA Ligase', Biochemistry 17: Kornberg, A., “Reversible Enzymatic Sysnthesis of 2069-2076 (1978). Diphosphopyridine nucleotide and inorganic pyrophos Middleton et al., “Synthesis and Purification of Oligo phate”, J Biol. Chem. 182: 779-793 (1950). nucleotides Using T4 RNA Ligase and Reverse-Phase Kaplan et al., “Enzymatic Deamination of Adenosine Chromatography', Analytical Biochemistry 144: Derivatives, J. Biol. Chem. 194: 579–591 (1952). 110-117 (1985). Bartkiewicz et al., “Nucleotide pyrophosphatase from Soltis et al., “Independent Locations of Kinase and potato tubers”, Eur. J. Biochem. 143: 419–426 (1984). 3'-Phosphatase Activities on T4 Polynucleotide Ki Rand et al., “Sequence and cloning of bacteriophage T4 nase', J. Biol. Chem. 257: 11340-11345 (1982). gene 63 encoding RNA ligase and tail fibre attachment Apostol et al., “Deletion Analysis of a Multifunctional activities’, The EMBO Journal 3: 397-402 (1984). yeast tRNA Ligase Polypeptide', J. Biol. Chem/ 266: Heaphy et al., “Effect of Single Amino Acid Changes in 7445-7455 (1991). the Region of the Adenylation Site of T4 RNA Igase', Becker et al., “The Enzymatic Cleavage of Phosphate Biochemistry 26: 1688-1696 (1987). Termini from Polynucleotides, J. Biol. Chern. 242: Lowe et al., “Molecular cloning and expression of a 936-950 (1967). cDNA encoding the membrane-associated rat intestinal Greer et al., “RNA Ligase in Bacteria: Formation of a alkaline phosphatase', Biochem. Biophys. Acta 1037: 2',5' Linkage by an E. coli Extract', Cell 33: 899-906 170–177 (1990). (1983). Chang et al., “Molecular Biology of Terminal Transfer Schwartz et al., “Enzymatic Mechanism of an RNA ase', CRC Crit. Rev. Biochem. 21: 27-52. Ligase from Wheat Germ', J. Biol. Chem. 258: Razzell et al., “Studies on Polynucleotides: III. Enzy 8374-8383 (1983). matic Degradation. Substrate Specificity and Properties Beabealashvilli, et al., "Nucleoside 5'-triphosphates of Snake Venom Phosphodiesterase”, J. Biol. Chem. modified at sugar residues as substrates for calf thymus 234: 2105-2113 (1959). terminal deoxynucleotidyl transferase and for AMV Tessier et al., "Ligation of Single-Stranded Oligodeox reverse transcriptase', Biochim. Biophys. Acta 868: yribonucleotides by T4 RNA Ligase', Analytical Bio 136-144 (1986). chemistry 158: 171-178 (1986). Lehman et al., “The Deoxyribonucelases of Escherichia England et al., “Enzymatic Oligoribonucleotide Syn coli, J. Biol. Chem. 239: 2628–2636 (1964). U.S. Patent July 25, 1995 Sheet 1 of 7 5,436,143 UNCONTROLLED METHOD primer -- nucleotide l TrOnsferose enzyme incubcation primer + (primer-- 1) + (prime?--2) + (primer--3) +...etc. Purify (primer--) product from side products (primer--1) Repeat cycle until oligonucleotide synthesis is complete BLOCKED METHOD primer + blocked-nucleotide TrOnsferose enzyme incubation primer + (prime?--1)-blocked + blocked-nucleotide noCivotion/RemovOlof TOnsferOse Removal of Blocking group (chemical or enzymatic) primer + (primer--1) + nucleotide Purify (primer--) product from nucleotide and primer (primer--1) Repeat Cycle until oligonucleotide synthesis is complete FIGURE U.S. Patent July 25, 1995 Sheet 2 of 7 5,436,143 primer + App (d) Np (or ATP + 3',5'-ADP) l RNA ligase incubation, then heat inactivate primer-(d) Np + primer + App (d) Np + AMP Alkaline Phosphatase incubation, then heat inactivote primer + primer-(d)N + App(d) N + Adenosine + PO Repeat cycle until oligonucleotide synthesis is complete FIGURE 2 primer + App (d) Np (or ATP + 3',5'-ADP) l RNA ligase incubation, then heat inactivate primer + primer-(d) Np + App(d) Np + AMP Exonuclease -- Nucleotide Pyrophosphotose incubation (e.g. venom PDE I), then hedt inoctivote primer-(d) Np + 3',5'-(d) NDP + AMP + (d) NMP's l Alkaline Phosphatase incubation, then heat inactivate primer-(d) N + (deoxy)nucleosides + adenosine + PO Repeat cycle until oligonucleotide synthesis is complete FIGURE 3 U.S. Patent July 25, 1995 Sheet 3 of 7 5,436,143 pyrophosphole AP -H CICentne -- O O O O BASE N/p -o-1N O -/ \ prime?-p(d)Np / O 3 O + primer -- o-/ BASE AMP N:f " SP/ Yo O /Y-C) O O O (H) 3',5'-(d)NDP osN / " / NO O App(d)Np pyrophosphole Odenne -- O Ap \/ p d BASE Y\ o1'N O s/ BASE 7 d / N O primer-p(d)Np(dNp o CD -> -- primer -- Sp- /- c. M o (H) AMP \ BASE2 N p - o \ BASE2 d Nsk P 'N Kd of o o / o p(d)Np(d)Nip Appo)Npd)Nip FIGURE 4 U.S. Patent July 25, 1995 Sheet 4 of 7 5,436,143 5' m 3' synthesized oligonucleotide ligote to oligonucleotide N1 with RNA ligose 5 Ham- 3' Onned to oligonucleotide N2 3' - 5' 5 - 3' Odd reverse transcriptase + dNTP's 3 - 5' 5' m- 3. Cleave Complimentary oligodeoxyribonucleotide from N2 with nuclease, e.g. RNase 3 - 5' complimentary oligodeoxyribonucleotide product FIGURE 5 U.S. Patent July 25, 1995 Sheet 5 of 7 5,436,143 primer + App (d) Np (or ATP + 3',5'-(d) NDP) Pump through RNA Ligose Column, recirculote if necessory Heat Inactivation of troce RNA Ligase leaked into solution (if necessory) primer + primer-(d) Np + App (d) Np + AMP Pump through Exonuclease + Nucleotide Pyrophosphotose Column (e.g. snoke venom PDEI), recirculote if necessory Hedt Inoctivation of troCe Exonucleose Ond Nucleotide Pyrophosphatase leaked into Solution (if necessory) primer-(d) Np + 3',5'-(d) NDP + AMP + (d) NMP's Pump through Alkaline Phosphatase Column, recirculate if necessary Heat Inactivation of troce Alkaline Phosphotose leaked into solution (if necessary) primer-(d) N + (deoxy) nucleosides + adenosine + PO Repeat cycle until oligonucleotide synthesis is Complete FIGURE 6 U.S. Patent July 25, 1995 Sheet 6 of 7 5,436,143 FIGURE 7 U.S. Patent July 25, 1995 Sheet 7 of 7 5,436,143 FIGURE 8A FIGURE 8B 5,436,143 1. 2 and the blocked nucleotide App(d)Np (or ATP--3',5'- METHOD FOR ENZYMATC SYNTHESS OF (d)NDP) have been used to make oligoribonucleotides OLGONUCLEOTIDES and oligodeoxyribonucleotides.