Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes.

Silver nanoassemblies constructed from boranephosphonate DNA.

Spatially selective deposition of metal onto complex DNA assemblies is a promising approach for the preparation of metallic nanostructures with features that are smaller than what can be produced by top-down lithographic techniques. We have recently reported the ability of 2'-deoxyoligonucleotides containing boranephosphonate linkages (bpDNA) to reduce AuCl4(-), Ag(+), and PtCl4(2-) ions to the corresponding nanoparticles. Here we demonstrate incorporation of bpDNA oligomers into a two-dimensional DNA array comprised of tiles containing double crossover junctions.

Reduction of metal ions by boranephosphonate DNA.

Oligodeoxyribonucleotides bearing boranephosphonate linkages (bpDNA) were shown to reduce a number of metal ions and form nanoparticles through a novel reaction pathway that leads to phosphate diesters or phosphate triesters in water or alcohols respectively. The synthetic utility of this reaction was further demonstrated through the synthesis of oligodeoxyribonucleotides containing phosphate triester linkages. This new reactivity also makes bpDNA promising for use in construction of DNA templated metallic nanostructures.

The synthesis of di- and oligo-nucleotides containing a phosphorodithioate internucleotide linkage with one of the sulfur atoms in a 5'-bridging position.

A new type of internucleotide phosphorodithioate linkage is described, wherein one of the sulfur atoms occupies a 5'-bridging position. Representative dinucleotides possessing such a bond were synthesized by S-alkylation of nucleoside-3'-O-phosphorodithioates with 5'-halogeno-5'-deoxy-nucleosides. A fully protected dithymidylate containing internucleotide 5'-S-phosphorodithioate linkage was converted into a 3'-O-phosphoramidite derivative and employed for introduction of a modified dinucleotide into a predetermined position of the oligonucleotide sequence.

Bis(sulfonamide) isosters of mycophenolic adenine dinucleotide analogues: inhibition of inosine monophosphate dehydrogenase.

Synthesis of novel inhibitors of human IMP dehydrogenase is described. These inhibitors are isosteric methylenebis(sulfonamide) analogues 5-8 of earlier reported mycophenolic adenine methylenebis(phosphonate)s 1-3. The parent bis(phosphonate) 1 and its bis(sulfonamide) analogue 5 showed similar sub-micromolar inhibitory activity against IMPDH2 (K(i) approximately 0.