July 2017

Bio-Synthesis Newsletter - July 2017

What are Molecular Inversion Probes?

MIPsMolecular Inversion Probes (MIPs) are single-stranded DNA molecules. MIPs contain sequences on their ends complementary to two regions flanking the target sequence of up to several hundred base pairs (bp). MIPs allow for targeted resequencing of tens or hundreds of kilobases (KB) without the need for the preparation of genomic libraries. After hybridization to the target, gap-filling and ligation results in a circular DNA molecule. This circular DNA molecule now contains the target sequence together with adaptor and barcoding sequences for downstream analysis. Other applications of MIPs are large-scale human SNP genotyping, resequencing large sets of human exons, detection of low-frequency variants, analysis of copy-number variation (CNV), accurate genotyping of similar prologs, quantification of alternative splicing, and medically relevant gene pools.

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Anti-CRISPR proteins inhibit the SpyCas9 protein.

Anti CRISPR protein 2LW5Recently anti-CRISPR proteins were identified that inhibit Streptococcus pyrogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. CRISPR-Cas9 systems defend against phage infections in bacteria. The crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and the anti-CRISPR protein AcrllA4 showed that AcrlA2 and AcrllA4 interact with SpyCas9 in a sgRNA dependent manner. AcrllA4 inhibits SpyCas9 by binding to the PAM-interacting site in the PAM-interacting domain. The binding blocks the recognition of double-stranded DNA substrates by SpyCas9. Dong et al. suggest that this may allow developing “off-switch” tools for SpyCas9 to avoid or minimize off-target effects that cause unwanted genome edits. The hope is that this will make CRISPR-based gene editing more precise and safer as well.

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Do you need streptavidin-biotin conjugates?

Streptavidin biotinThe proteins avidin and streptavidin are used for the design of valuable tools and reagents for the detection and purification of biotinylated compounds such as carbohydrates, nucleic acids, peptides, proteins, or other macromolecules. Streptavidin- or avidin-biotin tetramer complexes are the basis for many important biotechnological applications. The stability of the streptavidin- and avidin tetramers increases after binding to biotin or biotin-conjugates. This feature allows for the design of sensitive assays using micro-titer formats. A variety of conjugates can be designed allowing the production of reagents useful for molecular imaging or other assay types. Also, chemical approaches, such as click-chemistry, allow the attachment of linker molecules useful for further conjugation to compounds with desired properties. Examples are the conjugation of streptavidin or biotin to nanodots, oligonucleotides, peptides or polymer scaffolds. 

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Branched Oligonucleotides for the study of splicing events!

U6-snRNA-complex1Branched Nucleic Acids are known to participate in splicing of pre-mRNA. The small nuclear RNAs, U2 and U6 snRNA, are thought to form an x-shaped phosphotriester structure. To investigate how these triester molecules exist under physiological conditions, synthetic phosphate-branched nucleic acids are needed. Covalently branched DNA molecules contain a single nucleotide connected to three or more different oligonucleotide strands. In 2004, Heinonen and Lönnberg described the synthesis of phosphate-branched oligonucleotides. The scientists used nucleoside 3’- and 5’-phosphorodiamides as building blocks for the solid-phase synthesis of these branched oligomers. Furthermore, branched oligonucleotides can also be synthesized as well-defined two- and three-dimensional oligonucleotide assemblies for the design of DNA rods, DNA nanostructures or as multiple branched DNA as useful tools in nanotechnology and molecular diagnostics. 

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