Selenium Atom-Specific Mutagenesis (SAM) for Crystallography, DNA Nanostructure Design, and Other Applications - DNA Nanotechnology: From Structure to Function - page 37

Pinto et al. [ 45 ] further improved the 4 0 -Se nucleoside synthesis by making
A/T/C/U utilizing the Pummerer rearrangement of a selenoxide as the key step.
This improvement offers us an efficient way to synthesize all 4 0 -Se building blocks
for 4 0 -Se-containing RNA and DNA synthesis. In order to further investigate the
biophysical property of the 4 0 -Se nucleotides in duplexes, Pinto and Damha et al.
synthesized oligonucleotides containing 4 0 -Se-ribothymidine modification. Their
thermal denaturing and CD experiments suggested that the 4 0 -Se-oligonucleotides
behave more like RNA in terms of thermal-binding affinity and hairpin structure [ 46 ]
in contrast with the observed DNA-like conformation at nucleoside level [ 44 ]. Re-
cently, the DNA building block 4 0 -selenothymidine has been synthesized [ 47 ], and
surprisingly, it adopts the same 2 0 -endo pucker as thymidine. The 4 0 -Se-nucleotide
with the interesting conformational change could be applicable as a functionalized
moiety in nucleic acid nanotechnology, aptamers, and siRNA.

3.3 Phosphate Backbone-Modified SeNAs

3.3.1 Chemical Synthesis of Phosphoroselenoate Nucleic Acid

Phosphoroselenoate nucleic acids contain selenium at the phosphate backbone,
especially selenium replacing the non-bridging oxygen atoms. It was synthesized
through the backbone oxidation with KSeCN [ 48 ] for antisense study but proved to
be unstable in vivo. Egli and coworkers applied the phosphoroselenoate DNAs in
nucleic acid crystallography [ 49 ] by replacing a non-bridging oxygen atom with a
selenium atom (Fig. 3.4 ). In this case, a phosphoroselenoate moiety was introduced
into a hexamer DNA oligonucleotide (5 0 - C p-Se GCGCG-3 0 ) 2 (PDB ID: 1VRO), and
this Z-form DNA structure was determined by MAD phasing and refined to 1.1 A
resolution. The structure superimposition between the Se-modified and native DNAs
does not show the obvious structure difference, indicating again that the selenium
modification does not cause significant alteration in the native structure. Later, the
PSe-DNA phasing strategy was also successfully applied to the crystal structure
determination of a homo-DNA (6 0 -CGAATTCG-4 0 ) 2 [ 50 ].
With the phosphoramidite chemistry and automate DNA/RNA synthesizer, the
synthesis of PSe-DNAs became quite straightforward. Selenization of the phosphate
backbone can be achieved by using KSeCN (potassium selenocyanate) solution
during the oxidation step. One of the limitations of this method is that the yield
drops significantly due to the strong oxidant used in each chain elongation cycle,
especially when the modification site is far away from 5 0 -end of the target sequence.
Another limitation is that only the two diastereoisomers of short synthesized
oligonucleotides can be effectively separated using anion-exchanging HPLC [ 51 ].
Besides potassium selenocyanate, there are several alternative selenizing reagents
available, such as [(iPrO) 2 P(S)Se] 2 (di-0,O-isopropyl phosphoroselenothioate)

[ 52 ], BTSe (3H-1,2-benzothiaselenol-3one) [ 53 ], SePPh3 (triphenylphosphine