Supporting Information: Fast, Copper-Free Click Chemistry, A Convenient Solid-Phase Approach to Oligonucleotide Conjugation

General experimental Analytical TLC was performed on precoated (250 μm) silica gel 60 F-254 plates from Merck. All plates were visualized by UV irradiation, and/or staining with 5% H2SO4 in ethanol followed by heating. Flash chromatography grade silica gel 60 (230-400 mesh) was obtained from Merck. Mass analysis was performed on an Ettan MALDI-TOF Pro from Amersham Biosciences or LASER-TOF LT3 from Scientific Analytical Instruments with 3- hydroxypicolinic acid or 2,’ 4’, 6’-trihydroxyacetophenone as matrix. The NMR spectra were obtained at 1H (300 MHz), 13C (75 MHz) and 31P (121 MHz) on a Bruker instrument at 25 ºC. Chemical shifts are reported in ppm downfield from TMS as standard. HPLC was carried out using a Gilson instrument equipped with a UV detector and a Nucleosil C18 column (4.0 × 250 mm) or Phenomenex Clarity. Fluorescence spectra were recorded on a Varian Cary Eclipse instrument. All other chemical agents were purchased from Aldrich Chemical Company unless otherwise noted

Metal free click chemistry on nucleosides and oligonucleotides

Chemoselective ligation of biologically significant moieties through azide alkyne Click Chemistry has recently received much attention1. The reaction is attractive in that it regioselectively affords stable triazole linked bioconjugated products under mild conditions. However, from the view point of the synthetic oligonucleotide chemist, a significant disadvantage is that the non-thermal reaction requires an in situ generated Cu (I) catalyst. Unwanted Cu (I) mediated chemistry, specifically oxidative degradation etc

A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3′–5′ and 5′–3′ strands by rotating a single active site

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michealis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5ʹ-deoxythymidine-5ʹ-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then after chain extension using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 ̶ -3.1 °C per phosphoroselenolate) when introduced at the 5ʹ-termini of A-form duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity