Chemistry Reference
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deprotection step is then followed by the coupling step for the next base until the end of
the cyclic synthesis process is reached. Subsequently, the oligonucleotide is released from
the solid support by the treatment with aqueous ammonia which not only cleaves the ester
bonds through which the strands are attached to the solid phase, but also quantitatively
removes all protecting groups sitting on the (natural) nucleobases and the phosphate back-
bone. Depending on the chosen protecting group strategy, the artificial nucleotides may
require special operations of deprotection to obtain the fully deprotected single strand.
This step is followed by a purification using chromatography on disposable reverse-phase
cartridges or by HPLC or gel electrophoresis. After an optional desalting step, the prod-
ucts should be thoroughly characterized by MALDI- or ESI-mass spectrometry and
HPLC to confirm sequence identity, purity and full removal of protecting groups. The
concentration of the single-strands in solution is then estimated by UV spectroscopy
(requiring one to know the molar extinction of all incorporated nucleobases, including the
artificial one).
9.3.5 Enzymatic Oligonucleotides Synthesis
Although artificial nucleosides can be incorporated into short oligonucleotides by
chemical synthesis as described above, the enzymatic incorporation into long DNA
strands following the concept of the polymerase chain reaction (PCR) is highly desirable
to obtain even longer strands containing artificial nucleosides and to investigate the impli-
cation of nucleoside modifications on biological molecules and living systems. Using
very tolerant or engineered polymerases, indeed this has been shown to be possible.
Therefore, the artificial nucleosides have to be turned into 5 0 -triphosphates, the natural
substrates for polymerases, instead of 5 0 -DMT-protected 3 0 -phosphoramidites [35].
9.4 Assembly and Analysis of Metal Base Pairs Inside the DNA
Double Helix
9.4.1 Strategies for Metal Incorporation
The synthesis, purification and determination of DNA concentration yield solutions of
the matching single strands containing the ligand-modified nucleosides in either strand.
Prior to the addition of the metal ions forming the metal base pair, the double strand may
usually be preformed by allowing both complementary single strands to hybridize. This
is achieved by slowly cooling a hot solution of both strands combined in a 1 : 1 ratio in a
buffered aqueous solution at high ionic strength. The chosen buffer should provide a
suitable pH for metal complexation by the artificial nucleosides (and must not contain
EDTA or any other chelating agents!), the electrolyte is usually NaCl but may be changed
to NaClO 4 or NaNO 3 , when Ag(I) or Hg(II) are the metal ions of choice. The DNA con-
centration is usually in the micromolar range.
Subsequently, the metal ion under investigation is added as an aqueous solution
(usually prepared from a salt containing a weakly coordinating anion such as sulfate,
perchlorate or nitrate), often followed by various spectroscopic methods in the form of a
titration experiment [20].
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