Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.

The T−A and C−G base pairing and stacking allow the formation of the stable DNA duplex structure for genetic information storage, transcription, and replication. To replace the oxygen of the nucleotide nucleobases with selenium for the studies of the base-pair recognition, the duplex stability, and the nuclei acid crystal structures, we have synthesized for the first time the 4-Se thymidine phosphoramidite and incorporated it into oligonucleotides via solid-phase synthesis with high coupling yield (99%). The Se modification on the nucleobase is relatively stable under the elevated temperature. Using the dUSe (2‘-Se-dU) to facilitate the crystallization, we have successfully crystallized the DNA containing the 4-Se−T substitution and determined its structure at 1.50 Å resolution. The UV-melting and X-ray crystal structure studies have indicated that the Se substitution on the nucleobase does not cause a significant structure perturbation, the large Se atom on the thymine can be successfully accommodated by the DNA duplex, and the Se-mediated hydrogen bond (longer than the usual hydrogen bond) is formed within the modified T−A base pair. In addition, the Se derivatization on the nucleobases further facilitates X-ray crystal structure determination of nucleic acids and their protein complexes via Se MAD phasing.

To investigate nucleic acid base pairing and stacking via atom-specific mutagenesis and crystallography, we have synthesized for the first time the 6-Se-deoxyguanosine phosphoramidite and incorporated it into DNAs via solid-phase synthesis with a coupling yield over 97%. We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm (ε = 2.3 × 104 M−1 cm−1), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature. Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H. The crystal structure determination and analysis reveal that the overall structures of the native and Se-modified nucleic acid duplexes are very similar, the selenium atom participates in a Se-mediated hydrogen bond (Se … H–N), and the SeG and C form a base pair similar to the natural G–C pair though the Se-modification causes the base-pair to shift (approximately 0.3 Å). Our biophysical and structural studies provide new insights into the nucleic acid flexibility, duplex recognition and stability. Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.

To investigate nucleic acids with selenium derivatization for crystallography, we report the first synthesis of 2′-methylseleno-thymidine phosphoramidite and its incorporation into DNAs and RNAs by solid-phase synthesis with over 99% coupling yield. The d(GT Se GTACAC) 2 crystal structure was also determined at 1.40 Å resolution using Se phasing, revealing that this Se derivatization did not cause significant structure perturbation, consistent with our UV-melting study. In addition, we observed that the Se modification largely facilitated the crystallization.Nucleic acids play key roles in genetic information storage, expression and translation, and have attracted intense research attention for a long time 1-4 . X-ray crystallography has been widely used to study structure and function of nucleic acid molecules. 5-6 To facilitate crystal structure determination of nucleic acids, especially using Multiwavelength Anomalous Dispersion (MAD) or Single-Wavelength Anomalous Dispersion (SAD) phasing, 7 Huang, Egli and coworkers were the first to develop the selenium derivatization strategy. 8-13 So far, the 2′-Se derivatization has been introduced to U and C by us, and to A and G by other groups. 9-15 Using the Se derivatization and phasing, research laboratories have already reported several crystal structures of DNA and RNA molecules, 10,14-16 including the Homo-DNA structure, otherwise the phasing of which proved extremely challenging. 16 Natural tRNAs and rRNAs are heavily modified, and over 100 different modifications have been observed. 17 Ribothymidine (rT) is a common modification in mature tRNAs and rRNAs. Though the ribothymidine is the invariable modification at position 54 of tRNAs of nearly all bacteria and enkarya, 17b its function in the TΨC loop has not been well understood. Since our previous study indicated that the Se derivatization at the 2′-positions of the nucleotides does not cause significant perturbation of RNAs, 9-11 the Se incorporation at the 2′-position of ribothymidine in tRNAs and rRNAs will help to obtain insight into the important rT modification. Furthermore, the Se incorporation at the same 2′-position of thymidine will help to study structure and function of DNA molecules. Therefore, there is an urgent need to synthesize 2′-Se-thymidine derivative, as an analog of both ribothymidine and thymidine for the RNA and DNA studies. We report here the first synthesis of the 2′-methylseleno-thymidine derivative and the corresponding phosphoramidite, its incorporation into DNAs and RNAs by solid-phase synthesis, and the UV melting and X-ray crystal structure studies of the Sederivatized DNAs and RNAs.The synthesis of the 2′-methylseleno-ribothymidine derivative (4) started from the glycosylation of the protected D-ribose (1) with thymine under the Vorburggen conditions. 18 Intermediate 3 was synthesized by minor modifications of the literature procedures. 19 In brief, due to the 2′-neighboring group effect, this glycosylation generated exclusively the Huang@gsu.edu. NIH Public Access A...

Keywords: Nucleophilic vinylic substitution (S N V) mechanism / Enol ethers / Alkoxymethylene compounds / Quinoxalines / Molecular calculationsTreatment of 6-aminoquinoxaline with β,β-diactivated alkoxymethylene derivatives gave the corresponding N-(quinoxalin-6-yl)enamines. A variant of the S N V reaction mechanism was proposed for substitution of the alkoxymethylene compounds, on the basis of the structures of the precursor