Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code

Floyd E. Romesberg1, Chengzhi Yu1, Shigeo Matsuda1, Allison A. Henry1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.5
DOI:  10.1002/0471142700.nc0105s10
Online Posting Date:  November, 2002
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Abstract

This unit presents protocols for the synthesis and characterization of nucleosides with unnatural bases in order to develop bases for the expansion of the genetic alphabet or for nonselective pairing opposite natural bases. Protocols describe the design, synthesis, and characterization of unnatural base pairs involving 1‐β‐D‐2‐deoxyribosyl‐N‐ and ‐C‐nucleosides. Determination of the thermodynamic and kinetic parameters of unnatural nucleosides is accomplished by incorporation into oligonucleotides and subsequent evaluation as described herein.

     
 
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Table of Contents

  • Basic Protocol 1: General Guidelines for Synthesis of Pyrimidine‐Like 1‐β‐D‐2‐Deoxyribosyl‐n‐Nucleosides
  • Basic Protocol 2: General Guidelines for Synthesis of Purine‐Like 1‐β‐D‐2‐Deoxyribosyl‐N‐Nucleosides
  • Basic Protocol 3: Synthesis of 3,5‐Dimethylphenyl‐C‐Nucleoside
  • Basic Protocol 4: Synthesis of 1,4‐Dimethylnaphthalene‐C‐Nucleoside
  • Alternate Protocol 1: Synthesis of 3‐Methyl‐2‐Naphthalene‐C‐Nucleoside
  • Basic Protocol 5: Purification of DNA Oligonucleotides
  • Basic Protocol 6: Determination of Thermodynamic Stability of Unnatural Base Pairs
  • Basic Protocol 7: Kinetic Analysis of Unnatural Base Pair Incorporation, Selectivity and Replicability
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: General Guidelines for Synthesis of Pyrimidine‐Like 1‐β‐D‐2‐Deoxyribosyl‐n‐Nucleosides

  Materials
  • Argon (see recipe)
  • Isocarbostyril (S.1; Aldrich) or pyrimidine of choice
  • Acetonitrile, anhydrous
  • N,O‐Bis(trimethylsilyl)acetamide (Aldrich)
  • Bis‐toluoyl‐protected chloroglycoside: 1‐α‐chloro‐3,5‐di‐O‐toluoyl‐2‐deoxyribofuranose (S.2; Berry & Associates; Takeshita et al., )
  • SnCl 4, anhydrous, freshly distilled in vacuo
  • Ethyl acetate
  • Hexanes
  • Ammonium molybdate solution (see recipe)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Saturated sodium chloride (NaCl) solution
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel, 200 to 400 mesh, 60 Å
  • Dichloromethane (CH 2Cl 2), freshly distilled from calcium hydride
  • Ethyl ether, anhydrous
  • Iodine monochloride (ICl; Aldrich; packaged under nitrogen in Sure/Seal vials)
  • Saturated sodium thiosulfate (Na 2S 2O 3) solution
  • Magnesium sulfate (MgSO 4), anhydrous
  • Triethylamine (TEA), freshly distilled from calcium hydride
  • Dichlorobis(triphenylphosphine) palladium(II) [(Ph 3P) 2PdCl 2] (Aldrich)
  • Copper(I) iodide (CuI)
  • Dry ice/ethyl ether bath (–100°C) and dry ice/acetone bath
  • Propyne
  • Sodium methoxide (Aldrich)
  • Methanol, anhydrous, distilled from magnesium turnings and stored over 3A molecular sieves
  • Ammonium chloride (NH 4Cl)
  • Pyridine, anhydrous, distilled and stored on NaOH protected from light
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Aldrich)
  • Diisopropylethylamine (DIPEA), freshly distilled from calcium hydride
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite (Aldrich)
  • Trimethyl phosphate
  • Proton‐Sponge (Aldrich)
  • Phosphorous oxychloride (POCl 3), freshly distilled
  • Tributylamine
  • Tetrabutylammonium pyrophosphate (TBAP), stored over Drierite
  • 1 M triethylammonium bicarbonate (TEAB), pH 7
  • 10‐, 25‐, and 100‐mL two‐neck round bottom flasks with 14/20 joints, oven‐dried
  • Rubber septa
  • Vacuum system (oil pump) with manifold and cold trap
  • Silica gel 60 F 254 alumina‐backed thin‐layer chromatography (TLC) plates (Fisher)
  • Heat gun
  • UV light source
  • 100‐, 250‐, and 500‐mL separatory funnels
  • 300‐mL round bottom flask
  • Cotton
  • Glass funnel
  • Rotary evaporator (Büchi) equipped with a dry ice condenser and a vacuum system
  • Heavy‐walled glass columns (1.5‐cm i.d.; 10‐, 15‐, and 20‐cm length) with glass adapters attached to compressed air or nitrogen source (see flash chromatography steps in appendix 3E)
  • Sea sand
  • Reflux condenser with 14/20 joint
  • 23‐ and 18‐G needles
  • 100‐mL three‐neck flask with 14/20 joints
  • Propyne inlet adapter (Aldrich, cat. no. Z41.577‐4)
  • Teflon caps for 14/20 joints
  • Speedvac evaporator (Savant)
  • High‐performance liquid chromatography (HPLC) system
  • Glass or disposable plastic syringes
  • 10‐mL test tubes
  • Oil bath with temperature controller
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and flash chromatography ( appendix 3E)
NOTE: Glass or disposable plastic syringes are generally used for addition of liquid reagents. The 10‐mL test tubes are used for collection of eluting fractions in column chromatography. Oil baths with temperature controllers are used for heating reactions as necessary.

Basic Protocol 2: General Guidelines for Synthesis of Purine‐Like 1‐β‐D‐2‐Deoxyribosyl‐N‐Nucleosides

  Materials
  • Argon (see recipe)
  • 5H‐Pyrrolo[2,3‐b]pyrazine or purine analog of choice
  • Acetonitrile, anhydrous
  • 60% sodium hydride (NaH; see recipe)
  • Bis‐toluoyl‐protected chloroglycoside: 1‐α‐chloro‐3,5‐di‐O‐toluoyl‐2‐deoxyribofuranose (S.2; Berry & Associates; Takeshita et al., )
  • Ethyl acetate
  • Ethyl ether
  • Saturated sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel, 200‐400 mesh, 60 Å
  • Hexanes
  • 10‐mL two‐neck round‐bottom flask with 14/20 joints, oven‐dried
  • Rubber septa
  • 18‐G needle
  • Cotton
  • Glass funnel
  • 1.5 × 30–cm heavy‐walled glass column with glass‐adapters attached to compressed air or nitrogen source (see flash chromatography steps in appendix 3E)
  • Sea sand
  • Silica gel 60 F 254 alumina‐backed thin‐layer chromatography (TLC) plates (Fischer)
  • Heat gun
  • UV light source
  • Rotary evaporator (Büchi) equipped with a dry ice condenser and a vacuum system
  • Additional reagents and equipment for purification and analysis (see protocol 1).

Basic Protocol 3: Synthesis of 3,5‐Dimethylphenyl‐C‐Nucleoside

  Materials
  • Argon (see recipe)
  • Magnesium metal turnings
  • Tetrahydrofuran (THF), anhydrous
  • 1‐Bromo‐3,5‐dimethylbenezene (S.9; Aldrich)
  • Bis‐toluoyl‐protected chloroglycoside: 1‐α‐chloro‐3,5‐di‐O‐toluoyl‐2‐deoxyribofuranose (S.2; Takeshita et al., )
  • Ethyl acetate
  • Saturated ammonium chloride (NH 4Cl) solution
  • Sodium sulfate, anhydrous
  • Silica gel, 200‐400 mesh, 60 Å
  • Hexanes
  • Ethyl ether, anhydrous
  • Methanol, anhydrous
  • Sodium methoxide (Aldrich or synthesized from sodium metal and methanol)
  • Dichloromethane (CH 2Cl 2), freshly distilled from calcium hydride
  • Pyridine, anhydrous
  • Triethylamine (TEA), freshly distilled from calcium hydride
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Aldrich)
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • 4‐Dimethylaminopyridine (DMAP; Aldrich)
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite (Aldrich)
  • Trimethyl phosphate (Aldrich)
  • Proton‐Sponge (Aldrich)
  • Phosphorus oxychloride (POCl 3)
  • Tributylamine
  • n‐Tetrabutylammonium pyrophosphate (TBAP; Sigma)
  • Dimethylformamide (DMF)
  • Triethylammonium bicarbonate (TEAB; Fluka)
  • Dimethylsulfoxide (DMSO)
  • Isopropanol
  • 25‐mL and 100‐mL two‐neck round‐bottom flasks with 14/20 joints, oven‐dried
  • Reflux condenser with 14/20 joint
  • Rubber septa
  • 18‐ and 23‐G needles
  • 125‐mL separatory funnel
  • Cotton
  • Glass funnel
  • Rotary evaporator (Büchi) equipped with a dry ice condenser and a vacuum system
  • Heavy‐walled glass columns (1.5‐cm i.d.; 10‐ and 15‐cm length) with glass adapters attached to compressed air or nitrogen source (see flash chromatography steps in appendix 3E)
  • Sea sand
  • 300‐mL round‐bottom flask
  • Filter paper
  • Lyophilizer (e.g., Labconco freeze‐dry system)
  • Additional reagents and equipment for purification and analysis (see protocol 1)

Basic Protocol 4: Synthesis of 1,4‐Dimethylnaphthalene‐C‐Nucleoside

  Materials
  • Argon (see recipe)
  • 2‐Bromo‐1,4‐dimethylnaphthalene (S.14; Aldrich; Sharma, )
  • Tetrahydrofuran (THF), anhydrous
  • n‐Butyllithium (Aldrich)
  • Cyclohexane
  • Protected aldehyde of sugar precursor (S.15; Eaton and Millican, )
  • Ethyl acetate
  • Saturated sodium bicarbonate (NaHCO 3) solution
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel, 200‐400 mesh, 60 Å
  • Hexane
  • Ethyl acetate
  • Pyridine, anhydrous
  • Triethylamine (TEA), freshly distilled from calcium
  • Methanesulfonyl chloride
  • Acetic acid, glacial
  • Methanol, anhydrous
  • Trifluoroacetic acid
  • Dichloromethane (CH 2Cl 2), freshly distilled from calcium hydride
  • 2,3‐Dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ; Aldrich)
  • 25‐mL, 50‐mL, and 100‐mL two‐neck round bottom flasks with 14/20 joints, oven‐dried
  • Rubber septa
  • 18‐G needles
  • Dry ice/acetone bath (−78°C)
  • Cotton
  • Glass funnel
  • Rotary evaporator (Büchi) equipped with a dry ice condenser and a vacuum system
  • Heavy‐walled glass columns (1.5‐cm i.d.; 10‐, 15‐, and 20‐cm length) with glass adapters attached to compressed air or nitrogen source (see flash chromatography steps in appendix 3E)
  • Sea sand
  • Additional reagents and equipment for purification and analysis (see protocol 1)
NOTE:n‐Butyllithium is flammable and sensitive to moisture. It should be handled carefully and quickly.

Alternate Protocol 1: Synthesis of 3‐Methyl‐2‐Naphthalene‐C‐Nucleoside

  • 2‐Bromo‐3‐methylnaphthalene (S.19; prepared according to Lambert et al., )
  • Protected aldehyde of sugar precursor (S.20, prepared according to Solomon and Hopkins, )

Basic Protocol 5: Purification of DNA Oligonucleotides

  Materials
  • CPG‐bound DNA oligonucleotide ( appendix 3C)
  • Concentrated ammonium hydroxide
  • 95% (v/v) formamide in 10 mM Tris⋅Cl, pH 8.5 ( appendix 2A), with and without 0.05% (w/v) xylene cyanol and bromphenol blue
  • TBE buffer (see appendix 2A)
  • Absolute ethanol, prechilled to –20°C
  • 5 M NaCl, ice cold
  • 80% ethanol, ice cold
  • 1.5‐mL screw‐cap vial
  • 60° and 80°C Dri‐baths (Thermolyne)
  • Speed‐Vac evaporator (Savant)
  • 50‐mL polypropylene centrifuge tubes
  • UV/Vis spectrophotometer
  • Electroelution unit (Schleicher and Schuell)
  • Silica gel–coated preparative TLC plate
  • UV lamp (hand held)
  • Refrigerated centrifuge and rotor appropriate for sample size
  • Additional reagents and equipment for denaturing PAGE (unit 10.4 and appendix 3B)

Basic Protocol 6: Determination of Thermodynamic Stability of Unnatural Base Pairs

  Materials
  • Complementary 13‐mer oligonucleotides (e.g., S.22 and S.23 in water; see protocol 6)
  • T m buffer (see recipe)
  • Variable‐temperature double‐beam spectrophotometer (Cary 300 BIO UV‐Vis spectrophotometer) with optically matched cuvettes

Basic Protocol 7: Kinetic Analysis of Unnatural Base Pair Incorporation, Selectivity and Replicability

  Materials
  • 4 µM DNA oligonucleotide primer (e.g., S.24; see protocol 6)
  • 10 U/µL T4 polynucleotide kinase and 10× buffer (New England Biolabs)
  • 10 mCi/mL [γ‐33P]ATP (2500 Ci/mmol)
  • QIAquick Nucleotide Removal Kit (Qiagen)
  • 1 µM DNA oligonucleotide template (e.g., S.26; see protocol 6)
  • 10 U/µL exonuclease‐free Klenow fragment (Amersham Pharmacia Biotech) and 10× random‐prime buffer (see recipe)
  • Unnatural triphosphates (see Basic Protocols protocol 11 to protocol 44 and protocol 5)
  • 100 mM stock of each natural dNTP (Amersham Pharmacia Biotech; sequencing grade)
  • Enzyme dilution buffer (see recipe)
  • Quench solution (see recipe)
  • 1× TBE buffer ( appendix 2A)
  • 37°, 25°, and 100°C Dri‐Baths (Thermolyne) with heating blocks that accommodate 1.5‐ or 0.5‐mL tubes
  • Thin putty knife
  • Geiger counter
  • Chromatography paper (Whatman 3MM CHR, 35 × 45 cm)
  • Gel dryer (Bio‐Rad Model 583)
  • Storage Phosphor Screen (Kodak or Molecular Dynamics, 35 × 43 cm)
  • PhosphorImager (e.g., STORM Model 860; Molecular Dynamics)
  • ImageQuant and compatible spreadsheet (MS Excel) and graphing (Kaleidagraph) software
  • Additional reagents and equipment for denaturing PAGE (unit 10.4 and appendix 3B)
NOTE: Use autoclaved deionized water for all reagents.
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Figures

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Literature Cited

Literature Cited
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   Chaudhuri, N.C., Ren, R.X.F., and Kool, E.T. 1997. C‐Nucleoside derived from simple aromatic hydrocarbons. Synlett. 341‐347.
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   Eaton, M.A.W. and Millican, T.A. 1988. New methodology for C‐nucleoside synthesis: Preparation of 1,2‐dideoxy‐1‐(3‐pyridyl)‐D‐ribofuranose. J. Chem. Soc. Perkin Trans. I. 545‐547.
   Fischer, E. and Helferich, B. 1914. Synthetische Glucoside der Purine. Chem. Ber. 47:210.
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   Hildbrand, S., Blaser, A., Parel, S.P., and Leumann, C.J. 1997. 5‐Substituted 2‐aminopyridine C‐nucleosides as protonated cytidine equivalents: Increasing efficiency and selectivity in DNA triple‐helix formation. J. Am. Chem. Soc. 119:5499‐5511.
   Horlacher, J., Hottiger, M., Podust, V.N., Hübscher, U., and Bennier, S.A. 1995. Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. Proc. Natl. Acad. Sci. U.S.A. 92:6329‐6333.
   Kazimierczuk, Z., Cottam, H.B., Revankar, G.R., and Robins, R.K. 1984. Synthesis of 2′‐deoxytubercidin, 2′‐deoxyadenosine, and related 2′‐deoxynucleosides via a novel direct stereospecific sodium salt glycosylation procedure. J. Am. Chem. Soc. 106:6379‐6382.
   Kornberg, A. and Baker, T.A. 1992. DNA Replication, 2nd ed. W.H. Freeman and Co., New York.
   Lambert, J.B., Fabricus, D.M., and Hoard, J.A. 1979. Bond localization approach to the carbon analog of the Claisen rearrangement. Thermolysis of 4‐aryl‐1‐butenes. J. Org. Chem. 44:1480‐1485.
   Lambert, J.B., Shurvell, H.F., Lightner, D.A., and Cooks, R.G. 1998. Organic Structural Spectroscopy. Prentice Hall, Englewood Cliffs, N.J.
   Lee, E.L., Wu, Y., Xia, G., Schultz, P.G., and Romesberg, F.E. 2001. Efforts toward expansion of the genetic alphabet: Replication of DNA with three base pairs. J. Am. Chem. Soc. 123:7439‐7440.
   Lutz, M.J., Held, H.A., Hottiger, M., Hübscher, U., and Benner, S.A. 1996. Differential discrimination of DNA polymerases for variants of the non‐standard nucleobase pair between xanthosine and 2,4‐diaminopyrimidine, two components of an expanded genetic alphabet. Nucl. Acids Res. 24:1308‐1313.
   Lutz, M.J., Horlacher, J., and Benner, S.A. 1998a. Recognition of 2′‐deoxyisoguanosine triphosphate by HIV‐reverse transcriptase and mammalian cellular DNA polymerases. Bioorg. Med. Chem. Letts. 8:499‐504.
   Lutz, M.J., Horlacher, J., and Benner, S.A. 1998b. Recognition of a non‐standard base pair by thermostable DNA polymerases. Bioorg. Med. Chem. Letts. 8:1149‐1152.
   McMinn, D.L., Ogawa, A.K., Wu, Y., Liu, J., Schultz, P.G., and Romesberg, F.E. 1999. Efforts toward expansion of the genetic alphabet: DNA polymerase recognition of a highly stable, self‐pairing hydrophobic base. J. Am. Chem. Soc. 121:11585‐11586.
   Niedballa, U. and Vorbrüggen, H. 1970. A general synthesis of pyrimidine nucleosides. Angew. Chem. Int. Ed. Engl. 9:461.
   Niedballa, U. and Vorbrüggen, H. 1974. A general synthesis of N‐glycosides. I. Synthesis of pyrimidine nucleosides. J. Org. Chem. 39:3654‐3660.
   Ogawa, A.K., Wu, Y., McMinn, D.L., Liu, J., Schultz, P.G., and Romesberg, F.E. 2000a. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. J. Am. Chem. Soc. 122:3274‐3287.
   Ogawa, A.K., Wu, Y., Berger, M., Schultz, P.G., and Romesberg, F.E. 2000b. Rational design of an unnatural base pair with increased kinetic selectivity. J. Am. Chem. Soc. 122:8803‐8804.
   Postema, M.H.D. 1992. Recent developments in the synthesis of C‐glycosides. Tetrahedron. 48:8545‐8599.
   Ren, R.X.F., Chaudhuri, N.C., Paris, P.L., Rumney, S., and Kool, E.T. 1996. Naphthalene, phenanthrene, and pyrene as DNA base analogues: Synthesis, structure, and fluorescence in DNA. J. Am. Chem. Soc. 118:7671‐7678.
   Roberts, C., Chaput, J.C., and Switzer, C. 1997a. Beyond guanine quartets: Cation‐induced formation of homogenous and chimeric DNA tetraplexes incorporating iso‐guanine and guanine. Chem. & Biol. 4:899‐908.
   Roberts, C., Bandaru, R., and Switzer, C. 1997b. Theoretical and experimental study of isoguanine and isocytosine: Base pairing in an expanded genetic system. J. Am. Chem. Soc. 119:4640‐4649.
   Robinson, H., Gao, Y.‐G., Bauer, C., Roberts, C., Switzer, C., and Wang, A.H. 1998. 2′‐Deoxyisoguanosine adopts more than one tautomer to form base pairs with thymidine observed by high‐resolution crystal structure analysis. Biochemistry. 37:10897‐10905.
   Sharma, P.K. 1993. A new synthesis of trans‐3,4‐dihydroxy‐anti‐1,2‐epoxy‐1,2,3,4‐tetrahydro‐7,12‐dimethylbenz[a]anthracene. Synth. Commun. 23:389‐394.
   Solomon, M.S. and Hopkins, P.B. 1993. Chemical synthesis and characterization of duplex DNA containing a new base pair: A nondisruptive, benzofused pyrimidine analog. J. Org. Chem. 58:2232‐2243.
   Takeshita, M., Chang, C.N., Johnson, F., Will, S., and Grollman, A.P. 1987. Oligodeoxynucleotides containing synthetic abasic sites. J. Biol. Chem. 262:10171‐10179.
   Vorbrüggen, H. and Ruh‐Pohlenz, C. 2001. Handbook of Nucleoside Synthesis. John Wiley & Sons, New York.
   Vorbrüggen, H., Krolikiewicz, K., and Bennua, B. 1981. Nucleoside synthesis with trimethylsilyl triflate and perchlorate as catalysts. Chem. Ber. 114:1234‐1235.
   Wu, Y., Ogawa, A.K., Berger, M., McMinn, D.L., Schultz, P.G., and Romesberg, F.E. 2000. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. J. Am. Chem. Soc. 122:7621‐7632.
Key References
   Berger, M., Wu, Y., Ogawa, A.K., McMinn, D.L., Schultz, P.G., and Romesberg, F.E. 2000. Universal bases for hybridization, replication and chain termination. Nucl. Acids Res. 28:2911‐2914.
  This paper describes the evaluation of a variety of unnatural bases as universal bases for hybridization and replication.
   Berger, M., Luzzi, S.D., Henry, A.A., and Romesberg, F.E. 2002. Stability and selectivity of unnatural DNA with five‐membered‐ring nucleobase analogues. J. Am. Chem. Soc. 124:1222‐1226.
  This paper describes the evaluation of 5‐membered ring nucleobase analogs.
   Lee et al., 2001 See above.
  This paper describes the replication of DNA containing the 7AI self‐pair with the Klewnow fragment/pol β binary polymerase system.
   Ogawa et al., 2000a See above.
  This paper describes the synthesis of the C‐nucleosides reported herein.
   Ogawa et al., 2000b See above.
  This paper describes the rational design of the 3MN:3MN self‐pair.
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