Characterization

Matrix-Assisted Laser Desorption Ionization Mass Analysis

Purified synthetic oligodeoxynucleotides are often analyzed by negative matrix-assisted laser desorption ionization (MALDI) mass spectrometry to confirm the modification.67'68

Spectroscopic Analysis

Electronic absorption can be used to determine DNA concentration. All the nucleobases (A, G, C, and T) possess extensive it electron systems, which give rise to intense n— 7t* transitions that are observed in the electronic absorption spectra at ~250 nm. Because of base interactions, the absorbance of an oligodeoxynucleotide is not the direct sum of the individual base absorbances. In fact, the absorption spectrum for an oligodeoxynucleotide is a consequence of base composition and base interactions. To obtain an accurate extinction coefficient of an oligodeoxynucleotide, nuclease PI (Pénicillium citrinum) is used to cleave the entire oligodeoxynucleotide to its nucleotide components and thus eliminate all the A, C, G, and T 7r-stacking interactions.46 Once the oligodeoxynucleotide extinction coefficient is known, the sample concentration can be determined from Beer's Law.

The procedure for enzyme digestion and concentration determination is as follows.

1. Redissolve the pellet of HPLC-purified oligodeoxynucleotide in 100 /xl of deionized water.

2. Remove 5 (il of the oligodeoxynucleotide solution and add this sample to a microcentrifuge tube containing 1 (i 1 of a nuclease PI solution (1 mg/ml, 20 m M NaOAc, pH 5.5) and 44 (il of sodium acetate buffer (20 m M, pH 5.5).

4. Cool the sample to room temperature, dilute 10 ¡j\ of the sample to 1 ml with water, and measure the UV-visible (UV-Vis) absorbance.

67 K. J. Wu, A. Steding, and C. H. Becker, Rapid Commun. Mass Spectrom. 7, 142 (1993).

68 M. L. Gross and L. K. Zhang, J. Am. Soc. Mass Spectrom. 11, 854 (2000).

5. Determine the concentration [A = sbc, where c is the concentration, A is the absorbance, s is the sum of the absorption coefficients (A = 15,300 cm2 mol-1, C = 7400 cm2 moP1, G = 11,800 cm2 mol"', and T = 9300 cm2 mol-1), and b is the path length].

Melting Temperature

Electronic absorption spectroscopy can be used to monitor the denaturation and renaturation of double-stranded DNA. On denaturation, double-stranded DNA separates into single-stranded DNAs, with an absorption increase of approximately 40%. This increase in absorbance occurs over a narrow temperature range, with a sigmoidal curve indicating that the denaturation process is a cooperative event in which the collapse of one section of the structure destabilizes the remainder. The melting temperature (Tm) is defined as the temperature at which the maximum absorbance change (dA/dT) occurs. Longer DNA duplexes have higher melting temperatures and more stable secondary structures. The DNA sequence also influences the melting temperature, as a duplex possessing a large number of GC pairs will have a higher melting temperature compared with a duplex rich in AT pairs. The ionic strength of the solution also is known to affect the melting temperature. The melting temperature of labeled oligodeoxynucleotide duplexes is also dependent on the location of the redox probes on DNA and the interaction between the probe and DNA structure. The Tm can be, for example, higher if the probe is intercalated or lower if it is tethered to the base.

To form a duplex from two complementary strands, combine equal amounts of DNA strands in buffer solution (5 mM NaH2P04,50 mM NaCl, pH 7) and heat the resulting solution at 80° for 2-3 min, and then slowly cool the sample to room temperature. The melting curve profile is obtained with the following parameters on a Hewlett-Packard UV-Vis spectrophotometer: monitoring wavelength, 254 nm; temperature range, 20-70°; rate of temperature change, 0.5°/min.

Circular Dichroism Spectroscopy

Dichroism is the phenomenon in which light absorption changes for different directions of polarization. Circular dichroism (CD) refers to the absorption of the two different types of circularly polarized light.69 Nucleic acids have an intrinsic asymmetry because a chiral sugar is present within the structure. The formation of double-stranded oligodeoxynucleotides into a helical structure presents an asymmetry, which gives rise to strong interactions between the chromophore bases, generating an intense CD spectrum. As a result, CD spectra provide information about DNA secondary structure as A-form and B-form DNA (10.4 and 10.2), and Z-form DNA possess unique CD signatures. For example, the CD spectrum of

69 w. C. Johnson, "CD of Nucleic Acids." VCH, New York, 1994.

B-form (10.4) DNA contains positive CD features at 280 nm and 190 nm, and a negative feature at 240 nm.

Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) is another valuable technique to characterize the DNA melting transition. During the melting process, DNA strands change from a fully duplexed state to a fully separated single-strand state, and heat is absorbed as a result of this transition. Likewise, during hybridization, heat is generated. This absolute amount of heat change presents a direct way to determine the thermal stability of a labeled DNA duplex, and allows for the comparison of this DSC model-independent enthalpy determination with the model-dependent optical experiment method.70,71 In conventional DSC, the difference in heat flow between a sample and an inert reference is measured as a function of time and temperature. During the experiment, the change in heat is recorded as the temperature is increased and the area under this curve corresponds to the transition enthalpy.

A Microcal DSC instrument with a cell size of 0.51 ml is used for the DNA thermal analysis experiments. In a typical experiment, both the sample and reference cells are filled with buffer (5 mM NaH2P04, 50 mM NaCl, pH 7) first and the cells are scanned from 10 to 80° repeatedly at 0.57min. Once reproducible up-and-down baseline scans are obtained, the double-stranded oligodeoxynucleotide solution (35 fiM concentration) is loaded in the sample cell, leaving the reference cell unchanged. Again, the sample and control chambers are scanned from low to high temperature at a constant rate of 0.5°/min. The resulting curve is subtracted from the baseline and then normalized to concentration to afford the corrected heat capacity-versus-temperature curve. Finally, the area underneath this curve is calculated, and this enthalpy change can be used to compare the stability of labeled versus unlabeled DNA duplexes.

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