Synthesis of Long DNA-Based Nanowires

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 749)

Abstract

Here we describe novel procedures for production of DNA-based nanowires. This include synthesis and characterization of the one-to-one double-helical complex of poly(dG)–poly(dC), triple-helical poly(dG)–poly(dG)–poly(dC) and G4-DNA, which is a quadruple-helical form of DNA. All these types of DNA-based molecules were synthesized enzymatically using Klenow exo fragment of DNA Polymerase I. All the above types of nanowires are characterized by a narrow-size distribution of molecules. The contour length of the molecules can be varied from tens to hundreds of nanometers. These structures possess improved conductive and mechanical properties with respect to a canonical random-sequenced DNA and can possibly be used as wire-like conducting or semiconducting nanostructures in the field of nanoelectronics.

Key words

DNA nanowires Enzymatic synthesis Klenow exo Poly(dG)–poly(dC) G4-DNA Triplex DNA 

1 Introduction

The DNA molecule is an attractive candidate to wire electrons over long molecular distances. Charge migration along DNA molecules has been the subject of scientific interest for many years. It is currently accepted by the scientific community that a native, random sequence DNA is not a good electrical molecular wire, due to its apparent poor intrinsic conductivity. It has been demonstrated that uniform DNA comprising repeating sequences improves conduction properties. Recent experimental demonstration of the conducting behavior in short poly(dG)–poly(dC) DNA oligomers (1, 2) and the results of theoretical calculations show that poly(dG)–poly(dC), a homopolymer consisting of a pair of poly(dC) and poly(dG) chains, exhibits better conductance than poly(dA)–poly(dT) homopolymer (3). This is mainly due to the fact that poly(dG)–poly(dC) provides better conditions for π overlap compared to poly(dA)–poly(dT). In addition, ­guanine, which present in a high quantity in the former DNA is ­characterized by lowest ionization potential among DNA bases, thus promoting charge migration through the DNA (4). These facts emphasize the importance of guanine-rich DNA-based ­molecules as possible candidates for molecular electronics applications. The following guanine-rich DNA-based structures, whose syntheses is described here, may offer the desired electrical conduction properties (1) double-stranded poly(dG)–poly(dC), (2) triple-stranded poly(dG-dG)–poly(dC), and (3) four-stranded G4-DNA.

A poly(dG)–poly(dC) is a double-stranded deoxyribopolynucleotide polymer, which consists of a pair of antiparallel poly(dC) and poly(dG) homopolymers. Commercial preparations of the DNA are available and were used by researchers in electrical conductivity studies (5, 6). We have demonstrated (7), however, that commercial preparations of poly(dG)–poly(dC) consists of long continuous poly(dC)-strand and relatively short poly(G) fragments, 500–1,500 bases long, associated with the C-strand, but not covalently connected to each other. The presence of the G-strand breaks along poly(dG)–poly(dC) must strongly reduce the ability of the polymer to conduct current and strongly limits the use of the ­molecules in nanoelectronics. The molecules prepared by our technique (7) lack the above disadvantages. The enzymatic synthesis, conducted as described below, yielded double-stranded poly(dG)–poly(dC) characterized by a well-defined length (up to 10 kb) and narrow-size distribution of molecules. The synthesized molecules composed of continuous dG- and dC-homopolymers of equal length lacking strand nicks. In addition, the poly(dG)–poly(dC) may comprise a functional group attached to 5′ ends of either one or both strands composing the DNA (7). The functional group may be a particular sequence of single-stranded DNA, fluorescent labels, thiol-groups, biotin moieties, and other groups. Thiols are known to interact specifically with gold (8, 9). Introduction of SH-groups at the ends of DNA was used to anchor the DNA fragments to flat gold surfaces (10). The ability to attach SH-groups to the 5′ ends of the strands thus provides a tool for the selective binding of long poly(dG)–poly(dC) polymers to gold surfaces and gold nanoelectrodes. This property is especially useful for application of the polymer in nanoelectronics.

Triple-stranded DNA structures have been a subject of research in the past 50 years (  for review, see Refs. 11, 12, 13). Most of these structures are composed of tens of triads. Long triple-stranded DNA, poly(dG-dG)–poly(dC) have been reported by us only recently (14). A poly(dG-dG)–poly(dC) is an intramolecular triplex, composed of continuous dG- and dC-homopolymers. The length of the dG-homopolymer is twice the length of the poly(dC), which enables the former strand to fold back to pair with the adjacent of poly(dG)–poly(dC) duplex. The length of the latter triplex structures can be varied from ten to hundreds of nanometers. We were able to obtain the triplex nanostructures characterized by very narrow-size distribution by applying the enzymatic method described below. As in the case of poly(dG)–poly(dC) molecules, various functional group including fluorescent labels, thiol-groups can be attached to 5′ ends of either one or both strands composing the triplex (14). We have demonstrated that the triplex molecules are stiffer and more resistant to mechanical deformation compared to random sequence DNA and poly(dG)–poly(dC) (14). This property, together with the ability to functionalize the synthesized triplex molecules is essential for their application as elements in nanodevices.

G4-DNA wire is a very stable molecule made of consecutive stacked arrangement of 4 guanine (G) bases (tetrads – a tetrad consists of 4 G bases). It is known for decades that G-rich DNA sequences containing runs of guanines (dG) can form G-quadruplex structures (  for review see Refs. 15, 16, 17). These structures, commonly named G4-DNA, are comprised of stacked tetrads; each of the tetrads arises from the planar association of four guanines by Hoogsteen hydrogen bonding. Most of the studies have been performed using short (16–32 bases) G-rich telomeric oligonucleotides (18, 19). Short G-rich oligonucleotides were shown to assemble spontaneously into long molecular wires in the presence of proper monovalent cations (20, 21). These wires are very polymorphic, and constructed of short oligomers, resulting in nonuniform polymers with gaps (noncovalently bonded backbone) between G-rich oligonucleotide fragments along the formed wires (20, 21). Guanine tetrads were proposed as building blocks of molecular nanodevices (16, 17, 22). However, the above wires, formed by many short DNA segments, and containing many nicks are probably not good candidates for application as molecular nanowires. For the utilization of G4-DNA in nanoelectronics, long, persistent, homogenous populations of molecules are required. Only recently, we have reported a method (described below) for synthesis of novel long (hundreds of ­nanometers) continuous G-based nanostructures, composed of ­hundreds of stacked tetrads (23). These nanostructures are characterized by a narrow length distribution and contain no gaps in their backbone. We have also demonstrated that these wires are characterized by higher stability, resistance to heat treatment and higher charge polarizability, as compared to double-stranded DNA (24). These properties make these structures very promising for nanoelectronic applications.

Enzymatic synthesis of the long, di, tri, and tetra-stranded G-rich DNA-based nanostructures is described in detail below.

2 Materials

2.1 Preparation of (dG)12–(dC)12 Template–Primer

  1. 1.

    0.1 M NaOH solution: 4 g of NaOH. Add 1 L of fresh water purified by a Seralpur Pro 90 CN system (Merck Belgolabo, Overijse, Belgium), deionized water and filtered through 0.22 μm Millipore Express PLUS membrane filter, deionized/filtered water.

     
  2. 2.

    0.1 M NaOH containing 10% acetonitrile: 4 g of NaOH. Add 0.9 L of deionized/filtered water. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  3. 3.

    1 M phosphate buffer (pH 7.5): 136 g of KH2PO4. Add 700 mL of deionized water; adjust the pH to 7.5 with 1 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    20 mM Phosphate buffer (pH 7.5) containing 10% acetonitrile: to 880 mL of deionized water add 20 mL of 1 M Phosphate buffer (pH 7.5) and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  5. 5.

    250 mM Phosphate buffer (pH 7.5) containing 10% acetonitrile: to 650 mL of deionized water add 250 mL of 1 M Phosphate buffer (pH 7.5) and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  6. 6.

    Alkaline 1 M NaCl solution containing acetonitrile: 58 g of NaCl (Merck). Add 0.9 L of 0.1 M NaOH and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  7. 7.

    Glacial acetic acid 100%.

     
  8. 8.

    1 M Tris–acetate (pH 7.5): 121 g of Tris-base. Add 800 mL of deionized water; adjust the pH to 7.5 with glacial acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  9. 9.

    2 mM Tris–acetate (pH 7.5): to 998 mL of deionized/­filtered water add 2 mL of 1 M Tris–acetate (pH 7.5).

     
  10. 10.

    20 mM Tris–acetate (pH 7.5): to 980 mL of deionized/­filtered water add 20 mL of 1 M Tris–acetate (pH 7.5).

     
  11. 11.

    (dG)12 oligonucleotide from Alpha DNA (Montreal, Canada).

     
  12. 12.

    (dC)12 oligonucleotide from Alpha DNA (Montreal, Canada).

     
  13. 13.

    Dialysis tubing 10 mm (Sigma). Treat the tubing as follows: wash with running tap water for 3 h; treat with 0.3% (w/v) solution of sodium sulfide at 80°C for 1 min; wash with tap water for 2–5 min at 60°C; treat with 0.2% (v/v) solution of sulfuric acid for 10 min at room temperature; wash with tap water for 10–15 min. Store the tubing in 25% ethanol at 4°C. Rinse the tubing with running deionized/filtered water before use.

     
  14. 14.

    Ion-exchange PolyWax LP column (4.6  ×  200 mm, 5 μm, 1,000  Å) (Western Analytical Products).

     
  15. 15.

    Sephadex NAP-25 DNA-Grade column (15  ×  50 mm) (GE Healthcare).

     
  16. 16.

    Ion-exchange HiTrap Q HP column (1 mL) (GE Healthcare).

     
  17. 17.

    Agilent 1100 HPLC system with a photodiode array detector.

     
  18. 18.

    Eppendorf table centrifuge (model 5424).

     
  19. 19.

    Laboratory Freeze Dryer Christ Alpha 1–4 (Osterode am Harz, Germany).

     

2.2 Preparation of Thiol-End-Labeled (dG)12–(dC)12 Template–Primer [SH-(dG)12–(dC)12-SH]

  1. 1.

    0.1 M NaOH containing 10% acetonitrile: 4 g of NaOH. Add 0.9 L of deionized/filtered water. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  2. 2.

    5 M HCl solution: to 215 mL of deionized/filtered water add 285 mL of 32% HCL (Merck).

     
  3. 3.

    Alkaline 1 M NaCl solution containing acetonitrile: 58 g of NaCl. Add 0.9 L of 0.1 M NaOH and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  4. 4.

    1 M Tris–acetate (pH 7.5): 121 g of Tris-base. Add 800 mL of deionized water; adjust the pH to 7.5 with glacial acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  5. 5.

    2 mM Tris–acetate (pH 7.5): to 998 mL of deionized/­filtered water add 2 mL of 1 M Tris–acetate (pH 7.5).

     
  6. 6.

    20 mM Tris–acetate (pH 7.5): to 980 mL of deionized/­filtered water add 20 mL of 1 M Tris–acetate (pH 7.5).

     
  7. 7.

    0.4 M dl-dithiothreitol (DTT). 15.4 mg of DTT. Add 0.25 mL deionized/filtered water. Store at −18°C.

     
  8. 8.

    5′-(6-Mercapto-1-hexyl-phosphoric acid ester) of (dG)12 oligonucleotide, SH-(dG)12 from Alpha DNA (Montreal, Canada).

     
  9. 9.

    5′-(6-Mercapto-1-hexyl-phosphoric acid ester) of (dC)12 oligonucleotide, SH-(dC)12, from Alpha DNA (Montreal, Canada).

     
  10. 10.

    Dialysis tubing 10 mm (Sigma). Treat the tubing as follows: wash with running tap water for 3 h; treat with 0.3% (w/v) solution of sodium sulfide at 80°C for 1 min; wash with tap water for 2–5 min at 60°C; treat with 0.2% (v/v) solution of sulfuric acid for 10 min at room temperature; wash with tap water for 10–15 min. Store the tubing in 25% ethanol at 4°C. Rinse the tubing with running water and then with deionized/filtered water before use.

     
  11. 11.

    Ion-exchange PolyWax LP column (4.6  ×  200 mm, 5 μm, 1,000  Å), Western Analytical Products.

     
  12. 12.

    Sephadex NAP-25 DNA-Grade prepacked column (15  ×  50 mm) (GE Healthcare).

     
  13. 13.

    Ion-exchange HiTrap Q HP column (1 mL) (GE Healthcare).

     
  14. 14.

    Agilent 1100 HPLC system with a photodiode array detector.

     
  15. 15.

    Eppendorf table centrifuge (model 5424).

     
  16. 16.

    Laboratory Freeze Dryer Christ Alpha 1–4 (Osterode am Harz, Germany).

     

2.3 Synthesis of Poly(dG)–Poly(dC)

Enzymatic Synthesis of Poly(dG)–Poly(dC)

  1. 1.

    5 M KOH solution: 140 g of KOH. Add 0.5 L of deionized water.

     
  2. 2.

    1 M Phosphate buffer (pH 7.5): 136 g of KH2PO4. Add 700 mL of deionized water; adjust the pH to 7.5 with 5 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    1 M MgCl2: 20.3 g of MgCl2·6H2O. Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    1 M EDTA: 29.2 g of Titriplex II (Merck). Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  5. 5.

    0.4 M dl-dithiothreitol (DTT). 15.4 mg of DTT. Add 0.25 mL of deionized/filtered water. Store at −18°C.

     
  6. 6.

    100 mM dCTP. Dissolve 23.2 mg of dCTP (Sigma) in 0.5 mL of deionized/filtered H2O. Store at −18°C.

     
  7. 7.

    100 mM dGTP. Dissolve 25 mg of dGTP (Sigma) in 0.5 mL of deionized/filtered water. Store at −18°C.

     
  8. 8.

    10 μM (dG)12–(dC)12 prepared as described below (see Subheading 3.1).

     
  9. 9.

    Klenow exo (Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′  →  5′ exonuclease activity), 5 U/μL enzyme solution in glycerol from Fermentas (Lithuania). Store the solution at −18°C.

     
  10. 10.

    Dry bath incubator (MRC, Israel).

     

HPLC Purification of Synthesized Poly(dG)–Poly(dC)

  1. 1.

    1 M acetic acid solution: to 470 mL of deionized/filtered water add 30 mL of 100% acetic acid (Merck).

     
  2. 2.

    20 mM Tris–acetate (pH 8.0): 2.42 g of Tris-base (Fluka). Add 800 mL of deionized water; adjust the pH to 8.0 with 1 M acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    TSK-gel G-DNA-PW HPLC column (7.8  ×  300 mm) (TosoHaas, Japan).

     
  4. 4.

    Agilent 1100 HPLC system with a photodiode array detector.

     

2.4 Synthesis of Thiol-End-Labeled Poly(dG)–Poly(dC), SH-Poly(dG)–Poly(dC)-SH

Enzymatic Synthesis of SH-Poly(dG)–Poly(dC)-SH

  1. 1.

    5 M KOH solution: 140 g of KOH. Add 0.5 L of deionized water.

     
  2. 2.

    1 M Phosphate buffer (pH 7.5): 136 g of KH2PO4. Add 700 mL of deionized water; adjust the pH to 7.5 with 5 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    1 M MgCl2: 20.3 g of MgCl2·6H2O. Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    1 M EDTA: 29.2 g of Titriplex II (Merck). Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  5. 5.

    0.4 M dl-dithiothreitol (DTT). 15.4 mg of DTT (Sigma). Add 0.25 mL deionized/filtered water. Store at −18°C.

     
  6. 6.

    100 mM dCTP. Dissolve 23.2 mg of dCTP (Sigma) in 0.5 mL of deionized/filtered H2O. Store at −18°C.

     
  7. 7.

    100 mM dGTP. Dissolve 25 mg of dGTP (Sigma) in 0.5 mL of deionized/filtered water. Store at −18°C.

     
  8. 8.

    10 μM SH-(dG)12–(dC)12-SH prepared as described below (see Subheading 3.2).

     
  9. 9.

    Klenow exo (Klenow fragment of E. coli DNA polymerase I, lacking the 3′  →  5′ exonuclease activity), 5 U/μL enzyme solution in glycerol from Fermentas (Lithuania). Store the solution at −18°C.

     
  10. 10.

    Dry bath incubator (MRC, Israel).

     

HPLC Purification of Synthesized SH-Poly(dG)–Poly(dC)-SH

  1. 1.

    Glacial acetic acid 100%.

     
  2. 2.

    1 M Tris–acetate (pH 7.5): 121 g of Tris-base (Fluka). Add 800 mL of deionized water; adjust the pH to 7.5 with glacial acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    2 mM Tris–acetate (pH 7.5): to 998 mL of deionized/filtered water add 2 mL of 1 M Tris–acetate (pH 7.5).

     
  4. 4.

    20 mM Tris–acetate (pH 7.5): to 980 mL of deionized water add 20 mL of 1 M Tris–acetate (pH 7.5).

     
  5. 5.

    TSK-gel G-5000-PW HPLC column (7.8  ×  300 mm), TosoHaas, Japan.

     
  6. 6.

    Agilent 1100 HPLC system with a photodiode array detector.

     

2.5 Synthesis of Poly(dG-dG)–Poly(dC) Triplex

Poly(dG-dG)–Poly(dC) Triplex Synthesis

  1. 1.

    1 M KOH solution: 56 g of KOH. Add 1 L of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  2. 2.

    1 M Phosphate buffer (pH 7.5): 136 g of KH2PO4 (Merck). Add 700 mL of deionized water; adjust the pH to 7.5 with 1 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    1 M MgCl2: 20.3 g of MgCl2·6H2O. Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    1 M EDTA: 29.2 g of Titriplex II (Merck). Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  5. 5.

    0.4 M dl-dithiothreitol (DTT). 15.4 mg of DTT. Add 0.25 mL deionized/filtered water. Store at −18°C.

     
  6. 6.

    100 mM dGTP. Dissolve 25 mg of dGTP (Sigma) in 0.5 mL of deionized/filtered water. Store at −18°C.

     
  7. 7.

    1 mM (in base pairs) 500–1,000 base pairs poly(dG)–poly(dC) prepared as described in Subheading 3.3.

     
  8. 8.

    Klenow exo (Klenow fragment of E. coli DNA polymerase I, lacking the 3′  →  5′ exonuclease activity), 5 U/μL enzyme solution in glycerol from Fermentas (Lithuania). Store the solution at −18°C.

     
  9. 9.

    Dry bath incubator (MRC, Israel).

     

HPLC Purification of Synthesized Poly(dG-dG)–Poly(dC)

  1. 1.

    1 M acetic acid solution: to 470 mL of deionized/filtered water add 30 mL of 100% glacial acetic acid (Merck).

     
  2. 2.

    20 mM Tris–acetate (pH 7.5): 2.42 g of Tris-base. Add 800 mL of deionized water; adjust the pH to 7.5 with 1 M acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    TSK-gel G-DNA-PW HPLC column (7.8  ×  300 mm), TosoHaas, Japan.

     
  4. 4.

    Agilent 1100 HPLC system with a photodiode array detector.

     

2.6 Synthesis of G4 (Quadruple)-DNA

Purification of (dC)20

  1. 1.

    0.1 M NaOH solution: 4 g of NaOH. Add 1 L of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  2. 2.

    Glacial acetic acid 100% (Merck).

     
  3. 3.

    1 M Phosphate buffer (pH 7.5): 136 g of KH2PO4. Add 700 mL of deionized water; adjust the pH to 7.5 with 1 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    20 mM Phosphate buffer (pH 7.5) containing 10% acetonitrile; to 880 mL of deionized water add 20 mL of 1 M Phosphate buffer (pH 7.5) and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  5. 5.

    0.5 M Phosphate buffer (pH 7.5) containing 10% acetonitrile; to 0.4 L of deionized water add 0.5 L of 1 M Phosphate buffer (pH 7.5) and filter the solution through 0.22 μm Millipore Express PLUS membrane filter. Add 100 mL of acetonitrile (Bio Lab, HPLC-S Gradient grade).

     
  6. 6.

    1 M Tris–acetate (pH 7.5): 121 g of Tris-base (Fluka). Add 800 mL of deionized water; adjust the pH to 7.5 with glacial acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  7. 7.

    Tris–acetate (pH 7.5): to 998 mL of deionized/filtered water add 2 mL of 1 M Tris–acetate (pH 7.5).

     
  8. 8.

    10 μM (dG)12–(dC)12 prepared as described (see Subheading 3.1).

     
  9. 9.

    (dC)20 oligonucleotide from Alpha DNA (Montreal, Canada).

     
  10. 10.

    Dialysis tubing 10 mm (Sigma). Treat the tubing as follows: wash with running tap water for 3 h; treat with 0.3% (w/v) solution of sodium sulfide at 80°C for 1 min; wash with tap water for 2–5 min at 60°C; treat with 0.2% (v/v) solution of sulfuric acid for 10 min at room temperature; wash with tap water for 10–15 min. Store the tubing in 25% Ethanol at 4°C. Rinse the tubing with running deionized/filtered water before use.

     
  11. 11.

    Ion-exchange PolyWax LP column (4.6  ×  200 mm, 5 μm, 1,000  Å), Western Analytical Products.

     
  12. 12.

    Sephadex NAP-25 DNA-Grade prepacked column (15  ×  50 mm), GE Healthcare.

     
  13. 13.

    Ion-exchange HiTrap Q HP column (1 mL), GE Healthcare.

     
  14. 14.

    Agilent 1100 HPLC system with a photodiode array detector.

     
  15. 15.

    Eppendorf table centrifuge (model 5424).

     
  16. 16.

    Laboratory Freeze Dryer Christ Alpha 1–4 (Osterode am Harz, Germany).

     

Poly(dG)–n(dC)20 Synthesis

  1. 1.

    1 M KOH solution: 56 g of KOH. Add 1 L of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  2. 2.

    1 M Phosphate buffer (pH 7.5): 136 g of KH2PO4. Add 700 mL of deionized water; adjust the pH to 7.5 with 1 M KOH and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    20 mM Phosphate buffer (pH 7.5): to 98 mL of deionized water add 2 mL of 1 M Phosphate buffer (pH 7.5). Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  4. 4.

    1 M MgCl2: 20.3 g of MgCl2·6H2O. Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  5. 5.

    1 M EDTA: 29.2 g of Titriplex II (Merck). Add 100 mL of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  6. 6.

    0.4 M dl-dithiothreitol (DTT). 15.4 mg of DTT. Add 0.25 mL deionized/filtered water. Store at −18°C.

     
  7. 7.

    100 mM dGTP. Dissolve 25 mg of dGTP (Sigma) in 0.5 mL of deionized/filtered H2O. Store at −18°C.

     
  8. 8.

    10 μM (dG)12–(dC)12 template/primer prepared as described below (see Subheading 3.1).

     
  9. 9.

    HPLC-purified (dC)20 prepared as described below (see Subheading 3.6.1).

     
  10. 10.

    Klenow exo (Klenow fragment of E. coli DNA polymerase I, lacking the 3′  →  5′ exonuclease activity), 5 U/μL enzyme solution in glycerol from Fermentas (Lithuania). Store the solution at −18°C.

     
  11. 11.

    Dry bath incubator (MRC, Israel).

     

HPLC Purification of Synthesized Poly(dG)–n(dC)20

  1. 1.

    1 M acetic acid solution: to 470 mL of deionized/filtered water add 30 mL of 100% glacial acetic acid.

     
  2. 2.

    20 mM Tris–acetate (pH 7.5): 2.42 g of Tris-base. Add 800 mL of deionized water; adjust the pH to 7.5 with 1 M acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    TSK-gel DNA-G-DNA PW HPLC column (7.8  ×  300 mm) from TosoHaas, Japan.

     
  4. 4.

    Agilent 1100 HPLC system with a photodiode array detector.

     

Preparation of Poly(dG) Strands

  1. 1.

    1 M NaOH solution: 40 g of NaOH. Add 1 L of deionized water. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  2. 2.

    0.1 M NaOH: to 900 mL of deionized/filtered water add 100 mL of 1 M NaOH.

     
  3. 3.

    TSK-gel DNA-G-DNA PW HPLC column (7.8  ×  300 mm) from TosoHaas, Japan.

     
  4. 4.

    Agilent 1100 HPLC system with a photodiode array detector.

     

Preparation of G4-DNA

  1. 1.

    1 M acetic acid solution: to 470 mL of deionized/filtered water add 30 mL of 100% glacial acetic acid.

     
  2. 2.

    2 mM Tris–acetate (pH 8.0): 242 mg of Tris-base. Add 900 mL of deionized water; adjust the pH to 8.0 with 1 M acetic acid and add deionized water to 1 L. Filter the solution through 0.22 μm Millipore Express PLUS membrane filter.

     
  3. 3.

    Sephadex G-25 (NAP-5) prepacked DNA-Grade column (10  ×  30 mm), GE Healthcare.

     

3 Methods

3.1 Preparation of (dG)12–(dC)12 Template–Primer

Complete purification of (dG)12 and (dC)12 oligonucleotides comprising a (dG)12–(dC)12 template–primer from shorter or longer oligonucleotides is required (see Notes 1 and 2).

HPLC Purification of (dC)12

  1. 1.

    Transfer ∼1 mg of a dry oligonucleotide powder to 1.5 mL plastic tube.

     
  2. 2.

    Add 1 mL of deionized/filtered water.

     
  3. 3.

    Shake the sample and vortex vigorously for 2 min; incubate at room temperature for 30 min and vortex again.

     
  4. 4.

    Centrifuge the sample for 2 min at 5,000  ×  g at room temperature in order to get rid of insoluble compounds that might be present in the oligonucleotide preparation.

     
  5. 5.

    Transfer the entire supernatant to a new 1.5 mL plastic tube.

     
  6. 6.

    Connect an ion-exchange PolyWax LP column to the HPLC system.

     
  7. 7.

    Equilibrate the column with 50 mL of 20 mM Phosphate buffer (pH 7.5) containing 10% acetonitrile at a flow rate of 0.8 mL/min at room temperature.

     
  8. 8.

    Load 150 μL of the oligonucleotide sample at a flow rate of 0.8 mL/min. Do not overload the column; the large sample volume can significantly reduce the separation efficiency.

     
  9. 9.
    Elute the oligonucleotide in 10% acetonitrile with a linear Phosphate buffer gradient from 0.02 to 0.25 M for 30 min at a flow rate of 0.8 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm. The elution profile is shown in Fig. 1.
    Fig. 1.

    Purification of (dC)12 on a Poly WAX 300  Å column.

     
  10. 10.

    Collect the fraction containing (dC)12 eluted between 33.5 and 35 min (as indicated by the arrows in Fig. 1). Total volume of the fraction should be ∼1.0 mL.

     
  11. 11.

    Equilibrate the Sephadex G-25 DNA-Grade column with 30 mL of 2 mM Tris–acetate (pH 7.5) at room temperature.

     
  12. 12.

    Load 1 mL of the oligonucleotide sample obtained from the ion-exchange column (see step 10). Allow the sample to enter the column completely. Add 2.0 mL of Tris–acetate (pH 7.5). Allow the buffer to enter the column.

     
  13. 13.

    Place 2.0 mL plastic tube under the column; add 1.5 mL of 2 mM Tris–acetate (pH 7.5) and collect the eluant.

     
  14. 14.

    Transfer the sample into two 1.5 mL plastic tubes (1.0 mL per tube).

     
  15. 15.

    Freeze the sample in a dry ice/ethanol bath and lyophilize it to dryness. It takes approximately 15 h to completely lyophilize the sample.

     
  16. 16.

    Store the dry sample at −18°C.

     

HPLC Purification of (dG)12

  1. 1.

    Transfer ∼1 mg of a dry oligonucleotide powder to plastic 1.5 mL plastic tube.

     
  2. 2.

    Add 1 mL of 0.1 M NaOH.

     
  3. 3.

    Shake the sample and vortex vigorously for 2 min.

     
  4. 4.

    Centrifuge the sample for 2 min at 5,000  ×  g at room temperature in order to get rid of insoluble compounds that might be present in the oligonucleotide preparation.

     
  5. 5.

    Transfer the entire supernatant to a new 1.5 mL plastic tube.

     
  6. 6.

    Connect a HiTrap Q HP column to the HPLC system.

     
  7. 7.

    Equilibrate the column with 0.1 M NaOH containing 10% acetonitrile at a flow rate of 0.7 mL/min at room temperature.

     
  8. 8.

    Load 150 μL of the oligonucleotide sample at a flow rate of 0.7 mL/min.

     
  9. 9.
    Elute the oligonucleotide in 0.1 M NaOH containing 10% acetonitrile with a linear NaCl gradient from 0.5 to 1 M for 60 min at a flow rate of 0.7 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm. The elution profile is shown in Fig. 2.
    Fig. 2.

    Purification of (dG)12 on a HiTrap Q HP column.

     
  10. 10.

    Collect the fraction eluted between 35 and 37 min (as indicated by the arrows in Fig. 2). Total volume of the fraction should be ∼1.5 mL.

     
  11. 11.

    Equilibrate Sephadex G-25 DNA-Grade column with 30 mL of 2 mM Tris–acetate (pH 7.5) at room temperature.

     
  12. 12.

    Load 1.5 mL of the oligonucleotide sample obtained from the ion-exchange column (see step 10). Allow the sample to enter the column completely. Add 1.5 mL of 2 mM Tris–acetate (pH 7.5) buffer. Allow the buffer to enter the column.

     
  13. 13.

    Place 2.0 mL plastic tube under the column; add 2 mL of 2 mM Tris–acetate (pH 7.5) buffer and collect the eluant.

     
  14. 14.

    Transfer the solution into two 1.5 mL plastic tubes (1.0 mL per tube).

     
  15. 15.

    Freeze the sample in a dry ice/ethanol bath and lyophilize it to dryness. It takes approximately 15 h to completely lyophilize the sample.

     
  16. 16.

    Store the dry sample at −18°C.

     

Annealing of Purified (dC)12 and (dG)12

  1. 1.

    Dissolve HPLC-purified (dC)12 obtained as described in Subheading 3.1.1 in 200 μL of 0.1 M NaOH.

     
  2. 2.

    Withdraw 10 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 7.5).

     
  3. 3.

    Measure absorption of 100-times diluted sample at 260 nm.

     
  4. 4.

    Calculate the concentration of the oligonucleotide in the sample using an extinction coefficient of 90 mM−1 cm−1 at 260 nm. For example, the absorption of 0.9 corresponds to (dC)12 concentration in the stock solution of 1 mM.

     
  5. 5.

    Dissolve HPLC-purified (dG)12 obtained as described in Subheading 3.1.2 in 200 μL of 0.1 M NaOH.

     
  6. 6.

    Withdraw 10 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 7.5).

     
  7. 7.

    Measure absorption of 100-times diluted sample at 260 nm.

     
  8. 8.

    Calculate the concentration of the oligonucleotide in the sample using an extinction coefficient of 120 mM−1 cm−1 at 260 nm. For example, the absorption of 0.6 corresponds to (dG)12 concentrations in the stock solution of 0.5 mM.

     
  9. 9.

    Mix proper volumes of (dG)12 and (dC)12 samples (see above) to obtain a solution having equal final concentrations of both the above oligonucleotides. The final volume of the mixture should be 200–400 μL and the concentration of (dG)12–(dC)12 should be in the range of 10–30 μM.

     
  10. 10.

    Transfer the mixture to dialysis tubing and dialyze against 1 L of 20 mM Tris–acetate (pH 7.5) for 2 h at room temperature.

     
  11. 11.

    Withdraw 100 μL from the dialyzed sample and add to a quartz cuvette filled with 0.9 mL of 20 mM Tris–acetate (pH 7.5).

     
  12. 12.

    Measure absorption of tenfold diluted sample at 260 nm.

     
  13. 13.

    Calculate the concentration of the template–primer in the sample using an extinction coefficient of 177 mM−1 cm−1 at 260 nm. For example, the absorption of 0.177 corresponds to (dG)12–(dC)12 concentration in the stock solution of 10 μM.

     
  14. 14.

    Transfer the (dG)12–(dC)12 sample into several 0.5 mL plastic tubes (0.1 mL per tube).

     
  15. 15.

    Freeze the samples in a dry ice/ethanol bath and store at −18°C until ready to proceed with the DNA synthesis.

     

3.2 Preparation of SH-(dG)12–(dC)12-SH Template–Primer

Complete separation of SH-(dG)12 and SH-(dC)12 oligonucleotides, comprising a SH-(dG)12–(dC)12-SH template–primer, from shorter or longer oligonucleotides and oligonucleotides not containing thiol-groups is required.

HPLC Purification SH-(dC)12

  1. 1.

    Transfer ∼1 mg of a dry oligonucleotide powder to plastic 1.5 mL plastic tube.

     
  2. 2.

    Add 1 mL 20 mM Tris–acetate (pH 7.5).

     
  3. 3.

    Shake the sample and vortex vigorously for 2 min; incubate at room temperature for 30 min and vortex again.

     
  4. 4.

    Centrifuge the sample for 2 min at 5,000  ×  g at room temperature in order to get rid of insoluble compounds that might be present in the oligonucleotide preparation.

     
  5. 5.

    Transfer the entire supernatant to a new 1.5 mL plastic tube.

     
  6. 6.

    Add 25 μL of 0.4 M DTT.

     
  7. 7.

    Incubate the sample for 40 min at room temperature.

     
  8. 8.

    Connect an ion-exchange PolyWax LP column to the HPLC system.

     
  9. 9.

    Equilibrate the column with 20 mL of 2 mM Tris–acetate (pH 7.5) at a flow rate of 0.8 mL/min at room temperature.

     
  10. 10.

    Load 150 μL of the oligonucleotide sample at a flow rate of 0.8 mL/min. Do not overload the column; the large sample volume can significantly reduce the separation efficiency.

     
  11. 11.

    Elute the oligonucleotide with a linear NaCl gradient from 0 to 0.5 M for 60 min at a flow rate of 0.8 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  12. 12.

    Collect the fraction containing SH-(dC)12. Total volume of the fraction should be ∼1.5 mL.

     
  13. 13.

    Equilibrate Sephadex G-25 DNA-Grade column with 30 mL of 2 mM Tris–acetate (pH 7.5) at room temperature.

     
  14. 14.

    Load 1.5 mL of the oligonucleotide sample obtained from the ion-exchange column (see step 12). Allow the sample to enter the column completely. Add 1.5 mL of 2 mM Tris–acetate (pH 7.5). Allow the buffer to enter the column.

     
  15. 15.

    Place 2 mL plastic tube under the column; add 2.0 mL of Tris–acetate (pH 7.5) buffer and collect the eluant.

     
  16. 16.

    Transfer the solution into two 1.5 mL plastic tubes (1.0 mL per tube).

     
  17. 17.

    Freeze the sample in a dry ice/ethanol bath and lyophilize it to dryness. It takes approximately 15 h to completely lyophilize the sample.

     
  18. 18.

    Store the dry sample at −18°C.

     

HPLC Purification of SH-(dG)12

  1. 1.

    Transfer ∼1 mg of a dry oligonucleotide powder to plastic 1.5 mL plastic tube.

     
  2. 2.

    Add 1 mL of 0.1 M NaOH.

     
  3. 3.

    Shake the sample and vortex vigorously for 2 min.

     
  4. 4.

    Centrifuge the sample for 2 min at 5,000  ×  g at room temperature in order to get rid of insoluble compounds that might be present in the oligonucleotide preparation.

     
  5. 5.

    Transfer the entire supernatant to a new 1.5 mL plastic tube.

     
  6. 6.

    Add 25 μL of 0.4 M DTT.

     
  7. 7.

    Incubate the sample for 30 min at room temperature.

     
  8. 8.

    Connect an ion-exchange HiTrap Q HP column to the HPLC system.

     
  9. 9.

    Equilibrate the column with 0.1 M NaOH at a flow rate of 0.7 mL/min at room temperature.

     
  10. 10.

    Load 150 μL of the oligonucleotide sample at a flow rate of 0.7 mL/min.

     
  11. 11.

    Elute the oligonucleotide in 0.1 M NaOH with a linear NaCl gradient from 0.5 to 1 M for 200 min at a flow rate of 0.7 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  12. 12.

    Collect the fraction containing SH-(dG)12. Total volume of the fraction should be ∼3 mL.

     
  13. 13.

    Equilibrate Sephadex G-25 DNA-Grade column with 30 mL of 2 mM Tris–acetate (pH 7.5) at room temperature.

     
  14. 14.

    Load 3 mL of the oligonucleotide sample obtained from the ion-exchange column (see step 12). Allow the sample to enter the column completely.

     
  15. 15.

    Place 5 mL plastic tube under the column; add 3 mL of 2 mM Tris–acetate (pH 7.5) buffer and collect the eluant.

     
  16. 16.

    Transfer the solution into three 1.5 mL plastic tubes (1 mL per tube).

     
  17. 17.

    Freeze the sample in a dry ice/ethanol bath and lyophilize the sample to dryness. It takes approximately 15 h to completely lyophilize the sample.

     
  18. 18.

    Store the dry sample at −18°C.

     

Annealing of Purified SH-(dC)12 and SH-(dG)12

  1. 1.

    Dissolve HPLC-purified SH-(dC)12 obtained as described in Subheading 3.2.1 in 200 μL of 0.1 M NaOH.

     
  2. 2.

    Withdraw 10 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 7.5).

     
  3. 3.

    Measure absorption of 100-times diluted sample at 260 nm.

     
  4. 4.

    Calculate the concentration of the oligonucleotide in the sample using an extinction coefficient of 90 mM−1 cm−1 at 260 nm. For example, the absorption of 0.9 corresponds to SH-(dC)12 concentration in the stock solution of 1 mM.

     
  5. 5.

    Dissolve HPLC-purified SH-(dG)12 obtained as described in Subheading 3.2.2 in 200 μL of 0.1 M NaOH.

     
  6. 6.

    Withdraw 10 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 7.5).

     
  7. 7.

    Measure absorption of 100-times diluted sample at 260 nm.

     
  8. 8.

    Calculate the concentration of the oligonucleotide in the sample using an extinction coefficient of 120 mM−1 cm−1 at 260 nm. For example, the absorption of 0.6 corresponds to SH-(dG)12 concentration in the stock solution of 0.5 mM.

     
  9. 9.

    Mix proper volumes of SH-(dG)12 and SH-(dC)12 samples (see above) to obtain a solution having equal final concentrations of both the above oligonucleotides. The final volume of the ­mixture should be 200–400 μL and the concentration of SH-(dG)12–(dC)12-SH should be in the range of 10–30 μM.

     
  10. 10.

    Add 2.5 μL of 0.4 M DTT per each 100 μL the mixture and incubate at room temperature for 30 min.

     
  11. 11.

    Transfer the sample (200–400 μL) to dialysis tubing and dialyze against 100 mL of 20 mM Tris–acetate (pH 7.5) buffer containing 2 mM DTT for 1 h at room temperature. Change the dialysis buffer and continue dialysis for one more hour at room temperature.

     
  12. 12.

    Withdraw 50 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 7.5).

     
  13. 13.

    Measure absorption of 20 times diluted sample at 260 nm.

     
  14. 14.

    Calculate the concentration of the template–primer in the sample using an extinction coefficient of 177 mM−1 cm−1 at 260 nm. For example, the absorption of 0.177 corresponds to SH-(dG)12–(dC)12-SH concentration in the stock solution of 20 μM.

     
  15. 15.

    Transfer the SH-(dG)12–(dC)12-SH sample into several 0.5 mL plastic tubes (0.1 mL per tube).

     
  16. 16.

    Freeze the samples in a dry ice/ethanol bath and store at −18°C until ready to proceed with the DNA synthesis.

     

3.3 Preparation of Poly(dG)–Poly(dC)

Enzymatic Synthesis of Poly(dG)–Poly(dC)

The method described here is different from classical PCR-methods of DNA synthesis. It is based on the unique property of DNA Polymerase (Klenow exo fragment) to extend blunt-ended poly(dG)–poly(dC) molecules in the presence of dGTP and dCTP (see Note 3).
  1. 1.

    Prepare the mix for the DNA synthesis. Combine the following reagents in a 0.5 mL microcentrifuge plastic tube for each reaction: 85.25 μL of deionized/filtered water, 6 μL of 1 M Phosphate buffer (pH 7.5), 0.5 μL of 1 M MgCl2, 1.5 μL of 100 mM dCTP, 1.5 μL of 100 mM dGTP and 1.25 μL of 0.4 M DTT, for a total volume of 0.1 mL (you may scale up or down accordingly). Mix well by vortexing.

     
  2. 2.

    Add 2 μL of (dG)12–(dC)12 template/primer. Mix well by vortexing.

     
  3. 3.
    Add 2 μL of Klenow exo, mix well by vortexing and incubate the reaction at 37°C in an air dry bath for 1 h. One-hour incubation leads to synthesis of approximately 2,000 base pairs poly(dG)–poly(dC) molecules (see Fig. 3). You may change the amount of bases in the DNA accordingly by extending or reducing the incubation time.
    Fig. 3.

    Time course of poly(dG)–poly(dC) synthesis reaction. Polymerase extension assay was performed as described in Subheading 3.3.1 in the presence of 0.2 μM (dG)12–(dC)12 and 20 μg/ml of Klenow exo; the incubation was set at 37°C. Aliquots were withdrawn each 15 min for 2 h 15 min. (a) The reaction products were resolved on 1% agarose gel and stained with ethidium bromide. The marker bands of 1 kb DNA ladder (lane 1) are indicated to the left. Time-dependent products for 15, 30, 45, 60, 75, 90, 105, 120, and 135 min of the synthesis (lanes 2–10). (b) Dependence of the polymer length (in kb) estimated from (a) on the time of synthesis.

     
  4. 4.

    Add 2 μL of 1 M EDTA to terminate the reaction and vortex the sample.

     

HPLC Purification of Synthesized Poly(dG)–Poly(dC)

In order to completely separate synthesized Poly(dG)–Poly(dC) from nucleotides, the template–primer, Klenow exo, and other reaction components of the synthesis we recommend to use size-exclusion HPLC.
  1. 1.

    Connect TSK-gel G-DNA-PW HPLC column to the HPLC system (see Note 4).

     
  2. 2.

    Equilibrate the column with 20 mM Tris–acetate (pH 8.0) at a flow rate of 0.5 mL/min at room temperature.

     
  3. 3.

    Load 100 μL of poly(dG)–poly(dC) sample obtained as described above (Subheading 3.3.1) at a flow rate of 0.5 mL/min.

     
  4. 4.

    Elute the DNA in 20 mM Tris–acetate (pH 8.0) at a flow rate of 0.5 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  5. 5.

    Collect the DNA fraction from the column. Total volume of the fraction should be ∼1 mL.

     
  6. 6.

    Withdraw 100 μL from the DNA sample and add to a quartz cuvette filled with 1 mL of 20 mM Tris–acetate (pH 8.0).

     
  7. 7.

    Measure absorption of tenfold diluted sample at 260 nm.

     
  8. 8.

    Calculate the concentration of the DNA in the sample using an extinction coefficient of 14.8 mM−1 cm−1 at 260 nm for a GC pair. For example, the absorption of 0.148 corresponds to GC concentration in the stock solution of 0.1 mM.

     
  9. 9.

    Transfer the solution into several 0.5 mL plastic tubes (0.1 mL per tube) (see Note 5).

     
  10. 10.

    Freeze the DNA samples in a dry ice/ethanol bath and store at −18°C.

     

3.4 Preparation of SH-Poly(dG)–Poly(dC)-SH

The method enables to obtain homogeneous population of SH-poly(dG)–poly(dC)-SH characterized by high affinity to gold surfaces and electrodes.

Enzymatic Synthesis of SH-Poly(dG)–Poly(dC)-SH

  1. 1.

    Prepare the mix for the DNA synthesis. Combine the following reagents in a 0.5 mL microcentrifuge plastic tube for each reaction: 67 μL of deionized water, 6 μL of 1 M Phosphate buffer (pH 7.5), 0.5 μL of 1 M MgCl2, 1.5 μL of 100 mM dCTP, 1.5 μL of 100 mM dGTP and 1.5 μL of 0.4 M DTT, for a total volume of 0.1 mL (you may scale up or down accordingly). Mix well by vortexing.

     
  2. 2.

    Add 20 μL of SH-(dG)12–(dC)12-SH template/primer. Mix well by vortexing.

     
  3. 3.

    Add 2 μL of Klenow exo, mix well by vortexing and incubate the reaction at 37°C in an air dry bath for 1 h. One-hour incubation leads to synthesis of approximately 500 base pairs of SH-poly(dG)–poly(dC)-SH molecules. You may change the amount of bases in the DNA accordingly by extending or reducing the incubation time (see Note 6).

     
  4. 4.

    Add 2 μL of 1 M EDTA to terminate the reaction and vortex the sample.

     

HPLC Purification of Synthesized SH-Poly(dG)–Poly(dC)-SH

  1. 1.

    Connect TSK-gel G-DNA-PW HPLC column to the HPLC system.

     
  2. 2.

    Equilibrate the column with 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature.

     
  3. 3.

    Load 100 μL of SH-poly(dG)–poly(dC)-SH sample obtained as described above (Subheading 3.4.1) at a flow rate of 0.5 mL/min.

     
  4. 4.

    Elute the DNA in 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  5. 5.

    Collect the DNA fraction from the column. Total volume of the fraction should be ∼1 mL.

     
  6. 6.

    Withdraw 100 μL from the DNA sample and add to a quartz cuvette filled with 0.9 mL of 20 mM Tris–acetate (pH 7.5).

     
  7. 7.

    Measure absorption of tenfold diluted sample at 260 nm.

     
  8. 8.

    Calculate the concentration of the DNA in the sample using an extinction coefficient of 14.8 mM−1 cm−1 at 260 nm for a GC pair. For example, the absorption of 0.148 corresponds to GC concentration in the sample of 0.1 mM.

     
  9. 9.

    Transfer the solution into several 0.5 mL plastic tubes (0.1 mL per tube) (see Note 7).

     
  10. 10.

    Freeze the DNA samples in a dry ice/ethanol bath and store at −18°C.

     

3.5 Preparation of Poly(dG-dG)–Poly(dC)

The method of poly(dG-dG)–poly(dC) synthesis described here is based on the extension of the G-strand of the poly(dG)–poly(dC) by the Klenow exo fragment of DNA polymerase I, under conditions when only the G-strand is allowed to grow.

Enzymatic Synthesis of Poly(dG-dG)–Poly(dC)

  1. 1.

    Prepare the mix for the DNA synthesis. Combine the following reagents in a 0.5 mL microcentrifuge plastic tube for each reaction: 74.7 μL of deionized water, 6 μL of 1 M Phosphate buffer (pH 7.5), 0.33 μL of 1 M MgCl2, 0.5 μL of 100 mM dGTP, and 1.5 μL of 0.4 M DTT, for a total volume of 0.1 mL (you may scale up or down accordingly). Mix well by vortexing.

     
  2. 2.

    Add 15 μL of 1 mM (in base pairs) poly(dG)–poly(dC) ­solution. Mix well by vortexing.

     
  3. 3.

    Add 2 μL of Klenow exo, mix well by vortexing and incubate the reaction at 37°C in an air dry bath for 4 h.

     
  4. 4.

    Add 1 μL of 1 M EDTA to terminate the reaction and vortex the sample.

     

HPLC Purification of Synthesized Poly(dG-dG)–Poly(dC)

  1. 1.

    Connect TSK-gel G-DNA-PW HPLC column to the HPLC system.

     
  2. 2.

    Equilibrate the column with 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature.

     
  3. 3.

    Load 100 μL of poly(dG-dG)–poly(dC) sample obtained as described above (Subheading 3.5.1) at a flow rate of 0.5 mL/min.

     
  4. 4.

    Elute the triplex DNA in 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  5. 5.

    Collect the DNA fraction from the column. Total volume of the fraction should be ∼1 mL.

     
  6. 6.

    Withdraw 100 μL from the DNA sample and add to a quartz cuvette filled with 0.9 mL of 20 mM Tris–acetate (pH 7.5) buffer.

     
  7. 7.

    Measure absorption of tenfold diluted sample at 260 nm.

     
  8. 8.

    Calculate the concentration of the DNA in the sample using an extinction coefficient of approximately 20 M−1 cm−1 at 260 nm for a GGC triad. For example, the absorption of 0.2 corresponds to GGC concentration in the sample of 0.1 mM.

     
  9. 9.

    Transfer the solution into several 0.5 mL plastic tubes (0.1 mL per tube).

     
  10. 10.

    Freeze the DNA samples in a dry ice/ethanol bath and store at −18°C.

     

3.6 Preparation of G4 (Quadruple)-DNA

HPLC Purification of (dC)20

  1. 1.

    Transfer ∼1 mg of a dry oligonucleotide powder to plastic 1.5 mL plastic tube.

     
  2. 2.

    Add 1 mL of deionized/filtered water.

     
  3. 3.

    Shake the sample and vortex vigorously for 2 min; incubate at room temperature for 30 min and vortex again.

     
  4. 4.

    Centrifuge the sample for 2 min at 5,000  ×  g at room temperature in order to get rid of insoluble compounds that might be present in the oligonucleotide preparation.

     
  5. 5.

    Transfer the entire supernatant to a new 1.5 mL plastic tube.

     
  6. 6.

    Connect an ion-exchange PolyWax LP column to the HPLC system.

     
  7. 7.

    Equilibrate the column with 20 mL of 20 mM Phosphate buffer (pH 7.5), 10% acetonitrile, at a flow rate of 0.8 mL/min at room temperature.

     
  8. 8.

    Load 150 μL of the oligonucleotide sample at a flow rate of 0.8 mL/min. Do not overload the column; the large sample volume can significantly reduce the separation efficiency.

     
  9. 9.

    Elute the oligonucleotide with a linear gradient from 0.02 to 0.5 M Phosphate buffer (pH 7.5), 10% acetonitrile, for 60 min at a flow rate of 0.8 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  10. 10.

    Collect the DNA fraction from containing (dC)20. Total volume of the fraction should be ∼1 mL.

     
  11. 11.

    Equilibrate Sephadex NAP-25 DNA-Grade column with 30 mL of 2 mM Tris–acetate (pH 7.5) at room temperature.

     
  12. 12.

    Load 1 mL of the oligonucleotide sample obtained from the ion-exchange column (see step 10). Allow the sample to enter the column completely. Add 2 mL of 2 mM Tris–acetate (pH 7.5). Allow the buffer to enter the column.

     
  13. 13.

    Place 2 mL plastic tube under the column; add 2 mL of 2 mM Tris–acetate (pH 7.5) buffer and collect the eluant.

     
  14. 14.

    Transfer the solution into four 1.5 mL plastic tubes (0.5 mL per tube).

     
  15. 15.

    Freeze the sample in a dry ice/ethanol bath and lyophilize the sample to dryness. It takes approximately 15 h to completely lyophilize the sample.

     
  16. 16.

    Store the dry sample at −18°C.

     

Enzymatic Synthesis of Poly(dG)–n(dC)20

Klenow exo fragment of DNA Polymerase is capable of producing long double-stranded poly(dG)–n(dC)20 molecules composed of a long continuous dG-strand and relatively short dC-­oligonucleotides not covalently connected to each other in the presence of dGTP and (dC)20.
  1. 1.

    Prepare the mix for the DNA synthesis. Combine the ­following reagents in a 0.5 mL microcentrifuge plastic tube for each ­reaction: 78.5 μL of deionized water, 6 μL of 1 M Phosphate buffer (pH 7.5), 0.5 μL of 1 M MgCl2, 1.5 μL of 100 mM dGTP and 1.5 μL of 0.4 M DTT, for a total volume of 0.1 mL (you may scale up or down accordingly). Mix well by vortexing.

     
  2. 2.

    Dissolve HPLC-purified (dC)20 obtained as described in Subheading 3.6.1 in 200 μL of 20 mM Phosphate buffer (pH 7.5).

     
  3. 3.

    Withdraw 1 μL from the sample and add to a quartz cuvette filled with 1 mL of 20 mM Phosphate buffer (pH 7.5).

     
  4. 4.

    Measure absorption of 1,000-times diluted sample at 260 nm.

     
  5. 5.

    Calculate the concentration of the oligonucleotide in the sample using an extinction coefficient of 144 mM−1 cm−1 at 260 nm. For example, the absorption of 0.144 corresponds to (dC)20 concentration in the sample of 1 mM.

     
  6. 6.

    Add 10 μL of 1 mM (dC)20 to a final concentration of 100 μM.

     
  7. 7.

    Add 2 μL of (dG)12–(dC)12 template/primer. Mix well by pipetting.

     
  8. 8.

    Add 2 μL of Klenow exo, mix well by vortexing and incubate the reaction at 37°C in an air dry bath for 1 h. Two-hour incubation leads to synthesis of poly(dG)–n(dC)20 molecules composed of approximately 2,000 base-long G-strand. You may change the amount of bases in the DNA accordingly by extending or reducing the incubation time.

     
  9. 9.

    Add 2 μL of 1 M EDTA to terminate the reaction and vortex the sample.

     

HPLC Purification of Synthesized Poly(dG)–n(dC)20

  1. 1.

    Connect TSK-gel DNA-G-DNA PW HPLC column to the HPLC system.

     
  2. 2.

    Equilibrate the column with 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature.

     
  3. 3.

    Load 100 μL of poly(dG)–n(dC)20 sample obtained as described above (Subheading 3.6.2) at a flow rate of 0.5 mL/min.

     
  4. 4.

    Elute the DNA in 20 mM Tris–acetate (pH 7.5) at a flow rate of 0.5 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  5. 5.

    Collect the DNA fraction from the column. Total volume the fraction should be ∼1 mL.

     

Preparation of Poly(dG)

At pH higher than 12.5 the poly(dG)- and the (dC)20-fragments composing poly(dG)–n(dC)20 are separated from each other and are eluted separately from the HPLC column.
  1. 1.

    Connect TSK-gel G-DNA-PW HPLC column to the HPLC system.

     
  2. 2.

    Equilibrate the column with 0.1 M NaOH solution at a flow rate of 0.5 mL/min at room temperature.

     
  3. 3.

    Transfer 100 μL of poly(dG)–n(dC)20 sample obtained as described above (Subheading 3.6.3) to a 0.5 mL microcentrifuge plastic tube.

     
  4. 4.

    Add 15 μL of 1 M NaOH and incubate for 10 min at room temperature.

     
  5. 5.

    Load the sample onto the column at a flow rate of 0.5 mL/min.

     
  6. 6.

    Elute the DNA in 0.1 M NaOH at a flow rate of 0.5 mL/min at room temperature. Monitor the elution by measuring absorbance at 260 nm.

     
  7. 7.

    Collect a poly(dG) fraction eluted between 14 and 16 min. Total volume of the fraction should be ∼1 mL.

     

Preparation of G4-DNA

Folding of G-strands into G4-structures is taking place spontaneously upon pH reduction during chromatography of the alkaline strands solution on a Sephadex G-25 column.
  1. 1.

    Equilibrate a Sephadex G-25 column with 5 mL of 2 mM Tris–acetate (pH 8.0) at room temperature.

     
  2. 2.

    Load 0.5 mL of the alkaline G-strand sample obtained from the TSK-gel G-DNA-PW HPLC column (see Sub­heading 3.6.4). Allow the sample to enter the column ­completely. Add 0.2 mL of 2 mM Tris–acetate (pH 8.0). Allow the buffer to enter the column.

     
  3. 3.

    Place 1.5 mL plastic tube under the column; add 0.7 mL of 2 mM Tris–acetate (pH 8.0) buffer and collect the eluant.

     
  4. 4.

    Measure absorption of the sample at 260 nm.

     
  5. 5.

    Calculate the concentration of the DNA (in tetrads) in the sample using an extinction coefficient of 36 mM−1 cm−1 at 260 nm. For example, the absorption of 0.36 corresponds to 10 μM G4-DNA.

     
  6. 6.

    The sample can be stored for 2–3 days at 4°C. Longer storage is not recommended. Do not freeze the sample.

     

4 Notes

  1. 1.

    Complete purification of (dG)12, (dC)12, SH-(dG)12, SH-(dC)12 comprising a (dG)12–(dC)12 and SH-(dG)12–(dC)12-SH ­template–primers from shorter or longer oligonucleotides that are usually present in minor quantities in commercial preparations is required. If primed by nonpurified template–primers, the synthesis yields DNA molecules with large length variability.

     
  2. 2.

    Steps 8–13 in Subheadings 3.1.1 and 3.1.2 can be repeated several times in order to obtain larger quantities of purified oligonucleotides for a large-scale synthesis of the poly(dG)–poly(dC).

     
  3. 3.

    The protocol of poly(dG)–poly(dC) synthesis (see Subheading 3.3) can be adapted for synthesis of poly(dA)–poly(dT), a double-stranded polymer composed of poly(dA)- and poly (dT)-homopolymer strands of equal length.

     
  4. 4.

    If the length of the synthesized poly(dG)–poly(dC) is shorter than 1 Kbp, use TSK-gel G-5000-PW HPLC column (7.8  ×  300 mm, TosoHaas, Japan) instead of TSK-gel G-DNA-PW HPLC column (7.8  ×  300 mm, TosoHaas, Japan) for the HPLC purification of the DNA.

     
  5. 5.

    The G-containing structures are unstable at low pH and undergo acid hydrolysis. We thus recommend storage of the DNA samples at pH 8–8.5 at 4°C.

     
  6. 6.

    The rate of SH-poly(dG)–poly(dC)-SH synthesis is slower than that of non-thiolated poly(dG)–poly(dC) polymer. Relatively large quantities of the enzyme should therefore be added into the assay in order to obtain long SH-poly(dG)–poly(dC)-SH molecules.

     
  7. 7.

    Thiol-groups can undergo spontaneous oxidation to disulfides. We recommend to store SH-poly(dG)–poly(dC)-SH in the presence of 1 mM DTT to avoid disulfides formation. The molecules should be separated from DTT prior to deposition on gold surfaces. This can be done by passing the DNA sample through Sephadex G-25 DNA-Grade column equilibrated with 2 mM Tris–acetate (pH 7.5).

     

Notes

Acknowledgments

This work was supported by the EC through the contracts IST-2001-38951 (“DNA-Based Nanowires”) and FP6-029192 (“DNA-Based Nanodevices”).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biochemistry, The George S. Wise Faculty of Life SciencesTel Aviv UniversityRamat AvivIsrael

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