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Chemical and Enzymatic Methods for Preparing Circular Single‐Stranded DNAs

Amy M. Diegelman1,  Eric T. Kool1

1University of Rochester, Rochester, New York

Publication Name: 
Current Protocols in Nucleic Acid Chemistry
Unit Number: 
Unit 5.2
DOI: 
10.1002/0471142700.nc0502s00
Online Posting Date: 
May, 2001
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Abstract

Small circular oligonucleotides can be used for diagnostic, therapeutic, and laboratory purposes. These systems have gained considerable attention in recent years because they form unusually strong and specific complexes with RNA and DNA strands. Synthetic circular DNAs of 20 to 200 nucleotides can also serve as catalysts for amplified DNA and RNA synthesis by a rolling circle mechanism. This unit presents methods for synthesizing small circular oligonucleotides. These simple “one-pot” procedures are carried out using short DNA splints that hold the circle together until it is chemically or enzymatically ligated.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol: Double-Helical Splint Complex–Assisted Enzymatic Cyclization of Oligonucleotides Using T4 DNA Ligase
  • Alternate Protocol 1: Triple-Helical Splint Complex–Assisted Chemical Cyclization of Oligonucleotides Using Cyanogen Bromide
  • Alternate Protocol 2: Double-Helical Splint Complex–Assisted Reagent-Free Cyclization of Oligonucleotides Using a 3¢-Phosphorothioate Group and 5¢-Iodothymidine
  • Support Protocol 1: Automated Synthesis of Precursor Segments Containing 5¢-Phosphate for Ligation
  • Support Protocol 2: Automated Synthesis of Precursor Segments Containing 3¢-Phosphate for Ligation
  • Support Protocol 3: Automated Synthesis of Precursor Segments Containing 3¢-Phosphorothioate and 5¢-Iodothymidine for Ligation
  • Support Protocol 4: Deprotection of Precursor Segments
  • Support Protocol 5: Purification of Deprotected Precursor Segments (Optional)
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Double-Helical Splint Complex–Assisted Enzymatic Cyclization of Oligonucleotides Using T4 DNA Ligase

 Materials
  • 20 nt splint ssDNA(s)
  • Precursor segments containing 5¢-phosphate and 3¢-hydroxyl groups
  • Ultrapure (e.g., distilled, deionized) water
  • 2× ligation buffer: 20 mM MgCl2/100 mM Tris×Cl (pH 7.5)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • 25 mM and 100 mM adenosine triphosphate (ATP)
  • 400 U/µL T4 DNA ligase (New England Biolabs)
  • 1× TBE (appendix 2A)
  • 10% denaturing polyacrylamide gel mix (see recipe or purchase)
  • 10% (w/v) ammonium persulfate (APS)
  • TEMED or TMEDA (Life Technologies)
  • Formamide loading buffer (see recipe)
  • 0.2 N NaCl
  • 5× S1 nuclease buffer: 50 mM NaCl/50 mM NaOAc/5 mM ZnCl2, pH 4.6
  • 332 U/µL S1 nuclease (from Aspergillus oryzae; Amersham Pharmacia Biotech)
  • Stop solution (see recipe)
  • Stains-all dye solution (see recipe)
  • UV spectrophotometer
  • 1.5-mL microcentrifuge tubes and cap locks, autoclaved
  • 15-mL screw-top centrifuge tubes, autoclaved
  • 90°C heat block or thermal cycler
  • Glass wool
  • Dialysis tubing, MWCO 1000 (e.g., SpectraPor)
  • Gel electrophoresis equipment:
    •     Vertical (sequencing) gel stand
    •     2000-V power supply
    •     Glass plates: 13 × 15.5 cm, 13 × 16.5 cm, 6.5 × 15 cm, and 6.5 × 16 cm
    •     Gel combs: 1.5-mm-thick with 1.5-cm-wide wells and 0.4 mm thick with 5-mm-wide wells
    •     Spacers: 1.5 mm thick and 0.4 mm thick
  • UV shadow box or light source
  • Lyophilizer or SpeedVac
  • Saran Wrap (or other UV-transparent plastic wrap)
  • Razor blades, sterile
  • Glass stir rod
  • 50-mL filter tubes with 0.45-µm cellulose acetate filter (e.g., Spin-X II, Corning Costar)

Alternate Protocol 1: Triple-Helical Splint Complex–Assisted Chemical Cyclization of Oligonucleotides Using Cyanogen Bromide

 Additional Materials (also see Basic Protocol)
  • One purine-rich, triple-helical-forming splint ssDNA
  • Two triplex-forming precursor segments containing either 3¢- or 5¢-phosphates
  • 2× ligation buffer: 200 mM NiCl2/400 mM imidazole×HCl (pH 7.0)
  • Cyanogen bromide (BrCN; solid)

Alternate Protocol 2: Double-Helical Splint Complex–Assisted Reagent-Free Cyclization of Oligonucleotides Using a 3¢-Phosphorothioate Group and 5¢-Iodothymidine

 Additional Materials (also see Basic Protocol)
  • 20 nt splint ssDNAs
  • Precursor segments containing a 3¢-phosphorothioate and 5¢-iodothymidine
  • 2× ligation buffer: 20 mM MgCl2/20 mM Tris×acetate, pH 7.00

Support Protocol 1: Automated Synthesis of Precursor Segments Containing 5¢-Phosphate for Ligation

 Materials
  • DNA synthesis reagents for 0.2-µmol synthesis
  • Phosphorylating reagent (Chemical Phosphorylating Reagent, Glen Research)
  • DNA synthesizer with phosphoramidite chemistry

Support Protocol 2: Automated Synthesis of Precursor Segments Containing 3¢-Phosphate for Ligation

 Materials
  • DNA synthesis reagents for 0.2-µmol synthesis
  • Phosphorylating reagent (Chemical Phosphorylating Reagent, Glen Research)
  • DNA synthesizer with phosphoramidite chemistry

NOTE: This is analogous to addition of a 3¢ phosphorothioate in Support Protocol 3, but standard oxidizing (rather than sulfurizing) conditions are used to afford a 5¢-hydroxyl and 3¢-phosphate, rather than a 3¢-phosphorothioate, after deprotection (Support Protocol 4).

Support Protocol 3: Automated Synthesis of Precursor Segments Containing 3¢-Phosphorothioate and 5¢-Iodothymidine for Ligation

 Materials
  • DNA synthesizer with phosphoramidite chemistry
  • DNA synthesis reagents for 0.2-µmol synthesis
  • Phosphorylating reagent (S.1 in Fig. 5.2.8; Chemical Phosphorylating Reagent, Glen Research)
  • Sulfurizing reagent (S.3; Applied Biosystems)
  • Iodothymidine phosphoramidite (5¢-I-dT-CE phosphoramidite, Glen Research)

NOTE: Precursor segments will contain a 5¢-iodothymidine and a 3¢-phosphorothioate rather than 5¢-hydroxyl and 3¢-hydroxyl after deprotection (see Support Protocol 4).

Support Protocol 4: Deprotection of Precursor Segments

 Materials
  • Concentrated ammonium hydroxide
  • Pasteur pipets
  • Glass wool
  • 1.5-mL screw-cap vial

Support Protocol 5: Purification of Deprotected Precursor Segments (Optional)

 Materials
  • 10% denaturing polyacrylamide gel mix (see recipe or purchase)
  • 10% (w/v) ammonium persulfate (APS) in water
  • TEMED or TMEDA (Life Technologies)
  • 1× TBE (appendix 2A)
  • Ultrapure (e.g., distilled, deionized) water
  • Formamide loading buffer (recipe)
  • 0.2 N NaCl
  • Dialysis tubing, MWCO 1000 (e.g., SpectraPor)
  • 50-mL filter tubes with 0.45-µm cellulose acetate filter (e.g., Spin-X II, Corning Costar)
  • Gel electrophoresis equipment:
    •     Vertical (sequencing) gel stand
    •     2000-V power supply
    •     Glass plates: 13 × 15.5 cm and 13 × 16.5 cm
    •     2.5-mm-thick gel combs with 2.5-cm-wide wells
    •     2.5-mm-thick spacers
  • UV shadow box or light source
  • Lyophilizer or SpeedVac
  • Saran Wrap (or other UV-transparent plastic wrap)
  • Sterile razor blades
  • Glass stir rod
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 5.2.1
    Cyclization of ssDNAs requires a splint to assist in end joining. Representation of two types of splint complexes used for ligation: double-helical splint complex using conventional Watson-Crick hydrogen bonding, and triple-helical splint complex using both Watson-Crick and Hoogsteen hydrogen bonding.

    View Image
  • Figure 5.2.2
    Strategic planning for DNA circle synthesis using a decision tree. First decision is circle size. This decision will determine the number of precursor segments to use. The next decision involves analysis of sequence requirements and choice of a ligation method. AP, Alternate Protocol; BP, Basic Protocol; THSC, triple-helical splint complex.

    View Image
  • Figure 5.2.3
    Schematic representation of the steps in “one-pot” ligation for the enzymatic cyclization of ssDNAs by T4 DNA ligase. (A) Complementary splints 20-nt long are used to form double-helical splint complexes to juxtapose reactive 3¢-hydroxyl and 5¢-phosphate ends of linear ssDNAs for enzymatic ligation to a phosphodiester linkage (B) using T4 DNA ligase.

    View Image
  • Figure 5.2.4
    Representation of a typical cyclization gel. Lanes 1 to 3 represent crude synthesized DNAs used for ligation; banding underneath attests to their impurity. Lane 4 represents the first ligation-reaction mixture; the largest (slowest-migrating) species is the precircle, shown after isolation in lane 5. Lane 6 represents the second ligation (cyclization) and the possible products as confirmed by their migration with isolated species [lanes 7 to 9: 1.5× precircle (from ligation of 3 precursor segments in a linear fashion), circle and dimer]. Note the migration of the bands isolated in lanes 7 to 9 is typical but may differ slightly with different methods and sequences. Note: While the ligation mechanism selects for correct sequence precursor segments, it is not unusual to see less than full-length impurities (n – 1, n – 2, etc.), and these impurities are often eliminated by careful isolation of the desired band.

    View Image
  • Figure 5.2.5
    Representation of a typical characterization gel. Lanes 1 and 3 represent isolated precircle and circle controls. Lanes 2 and 4 represent precircle and circle cleavage by S1 nuclease, an endonuclease. As would be expected for endonuclease cleavage of a circle (lane 4), the initial cleavage produces a species that exhibits the same migration as the full-length cyclization precursor (precircle). Initial endonuclease cleavage of the linear precircle (see in lane 2 and also lane 4), by contrast, produces a continuous banding pattern.

    View Image
  • Figure 5.2.6
    Schematic representation of ligation steps for the chemical cyclization of ssDNAs using cyanogen bromide, BrCN. (A) A purine-rich ssDNA splint is used to form a triple-helical splint complex to juxtapose reactive 3¢-phosphate and 5¢-hydroxyl ends of linear DNAs for a simultaneous dimeric ligation to a phosphodiester linkage, (B), using BrCN. Note: For chemical ligation using BrCN, a 5¢-phosphate and 3¢-hydroxyl such as are used in Figure 5.2.3B are also suitable.

    View Image
  • Figure 5.2.7
    Schematic representation of “one-pot” autoligation steps for the reagent-free cyclization of ssDNAs. (A) Complementary splints are used to form double-helical splint complexes to juxtapose reactive 3¢-phosphorothioate and 5¢-iodothymidine ends of linear ssDNAs for autoligation via nucleophilic attack by phosphorothioate on iodothymidine. (B) Basic chemistry of autoligation.

    View Image
  • Figure 5.2.8
    Schematic representation of steps involved in incorporation of a 3¢-phosphorothioate group during DNA synthesis. First, a 3¢-phosphate is coupled to the base on the CPG bead, S.1. Addition of the next base, S.2, the 3¢ base of the desired sequence, proceeds with the trivalent phosphorus of this internucleotide bond converted to a phosphorothioate linkage using a sulfurizing reagent, S.3. Standard deprotection cleaves 3¢ to this phosphorothioate linkage, S.4, creating a 3¢-phosphorothioate group for coupling, S.5.

    View Image

Literature Cited

 Literature Cited
    Alazzouzi, E., Escaja, N., Grandas, A., and Pedroso, E. 1997. A straightforward solid-phase synthesis of cyclic oligodeoxyribonucleotides. Angew. Chem. Int. Ed. Engl. 36:1506-1508.
    Ashley, G.W. and Kushlan, D.M. 1991. Chemical synthesis of oligodeoxynucleotide dumbbells. Biochemistry 30:2927-2933.
    Borer, P.N. 1975. Optical properties of nucleic acids. In Handbook of Biochemistry and Molecular Biology, Vol.I, 3rd ed. (G.D. Fasman, ed.) p. 589. CRC Press, Boca Raton, Fla.
    Brown, S. 1997. Metal-recognition by repeating polypeptides. Nature Biotechnol. 15:269-272.
    Daubendiek, S.L. and Kool, E.T. 1997. Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles. Nature Biotechnol. 15:273-277.
    De Napoli, L., Galeone, A., Mayol, L., Messere, A., Montesarchio, D., and Piccialli, G. 1995. Automatic solid phase synthesis of cyclic oligonucleotides: A further improvement. Bioorg. Med. Chem. 3:1325-1329.
    Dolinnaya, N.G., Sokolova, N.I., Ashirbekova, D.T., and Shabarova, Z.A. 1991. The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids: Comparison with water-soluble carbodiimide. Nucl. Acids Res. 9:3067-3072.
    Dolinnaya, N.G., Blumenfeld, M., Merenkova, I.N., Oretskaya, T.S., Krynetskaya, N.F., Ivanovskaya, M.G., Vasseur, M., and Shabarova, Z.A. 1993. Oligonucleotide circularization by splint-directed chemical ligation. Nucl. Acids Res. 21:5403-5407.
    Fire, A. and Xu, S.Q. 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. U.S.A. 92:4641-4645.
    Herrlein, M.K. and Letsinger, R.L. 1994. Selective chemical autoligation on a double-stranded DNA splint. Nucl. Acids Res. 22:5076-5078.
    Herrlein, M.K., Nelson, J.S., and Letsinger, R.L. 1995. A covalent lock for self-assembled oligonucleotide conjugates. J. Am. Chem. Soc. 117:10151-10152.
    Ippel, J.H., Lanzotti, V., Galeone, A., Mayol, L., Van den Boogaart, J.E., Pikkemaat, J.A., and Altona, C. 1995. Slow conformational exchange in DNA minihairpin loops: A conformational study of the circular dumbbell d<pCGC-TT-GCG-TT>. Biopolymers 36:681-694.
    Kool, E.T. 1996. Circular oligonucleotides: New concepts in oligonucleotide design. Annu. Rev. Biophys. Biomol. Struct. 25:1-28.
    Liu, D., Daubendiek, S.L., Zillman, M.A., Ryan, K., and Kool, E.T. 1996. Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118:1587-1594.
    Nilsson, M., Malmgren, H., Samiotaki, M., Kwiatkowski, M., Chowdhary, B.P., and Landegren, U. 1994. Padlock probes: Circularizing oligonucleotides for localized DNA detection. Science 265:2085-2088.
    Nilsson, M., Krejci, K., Koch, J., Kwiatkowski, M., Gustavsson, P., and Landegren, U. 1997. Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nature Genet. 16:252-255.
    Ruben, E., Rumney, S. IV, Wang, S., and Kool, E.T. 1995. Convergent DNA synthesis: A non-enzymatic dimerization approach to circular oligodeoxynucleotides. Nucl. Acids Res. 23:3547-3553.
    Rumney, S.IV. and Kool, E.T. 1992. DNA recognition by hybrid oligoether-oligodeoxynucleotide macrocycles. Angew. Chem. Int. Ed. Engl. 31:1617-1619.
    Serwer, P. and Allen, J.L. 1984. Conformation of double-stranded DNA during agarose gel electrophoresis: Fractionation of linear and circular molecules with molecular weights between 3 × 106 and 26 × 106. Biochemistry 23:922-927.
    Wang, S. and Kool, E.T. 1994. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs. Nucl. Acids Res. 22:2326-2333.
    Xu, Y. and Kool, E.T. 1997. A novel 5¢-iodonucleoside allows efficient nonenzymatic ligation of single-stranded and duplex DNAs. Tetrahedron Lett. 38:5595-5598.
 Key References
    Alazzouzi et al., 1997. See above.

Reports a unique methodology for the solid-phase synthesis of small (<32 nt) ssDNA circles. No splint is needed, but yields are low for the larger circles in this size range.

    Dolinnaya et al., 1993. See above.

Reports the synthesis of medium-sized (~40 nt) ssDNA circles from a double-helical splint complex with cyanogen bromide. Certain sequence requirements for successful ligation are described.

    Ruben et al., 1995. See above.

Reports the cyanogen bromide–mediated synthesis of medium-sized (34- to 72-nt) ssDNA circles from two short segments using a triple-helical splint complex.

    Xu and Kool, 1997. See above.

Reports an autoligation method incorporating a novel 5¢-iodothymidine phosphoramidite (now commercially available) which possesses increased stability as compared to one previously described (Herrlein et al., 1995).

     
 
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