The Potential Use of Aptamers in The Process of Drug Development

Single-stranded nucleic acids can fold and create unique 3-dimensional structures when interacting with other molecules. The unique structure can achieve high specificity and affinity for the particular target. Synthetic oligonucleotide binding agents also known as aptamers are generated through the rational process of Systematic Evolution of Ligands by Exponential Enrichment (SELEX.) As this technology matures it shows increasing promise for use in the field of a therapeutic drug, drug discovery, development, and delivery, and this report seeks to detail how this technology may be applied.

Scale-Up of a Heck Alkenylation Reaction: Application to the Synthesis of an Amino-Modifier Nucleoside ‘Ruth Linker’

Ruth linker is a C5 pyrimidine modified nucleoside analogue widely utilized for the incorporation of a primary amine in a synthetic oligonucleotide. The increasing demand for non-radioactive labeling, detection of biomolecules, and assembly of COVID-19 test kits has triggered a need for scale-up of Ruth linker. Herein, an efficient protocol involving a palladium-catalyzed Heck alkenylation is described. The synthesis has been optimized with a goal of low catalyst concentration, column-free isolation, high product purity, reproducibility, and shorter reaction time. The scalability and utility of the process have been demonstrated successfully on a 100 g scale (starting material). Additionally, for scale-up of the Heck alkenylation protocol, 7-phospha-1,3,5-triaza-adamantanebutane sulfonate (PTABS) as the coordinating caged phosphine ligand was also synthesized on a multigram scale after careful optimization of the conditions.

CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.

CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different distinct libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.

Antisense Technology

Diseases are often connected on the expression of some disease causing gene which is important to produce that Protein. If the expression of this gene can be disputed then the disease can be cured. Antisense technology is a method of disputing the production of protein. It may be used to design some therapeutics for diseases in whose pathology is the production of protein plays a major role. Antisense technology is important tool in the inhibition of that particular gene expression. The principle behind it, is that the antisense nucleic acid sequence base pairs with its complementary sense RNA strand is inserted and prevents the RNA from translated into protein. The complementary nucleic acid sequence is either complementary synthetic oligonucleotide, often oligodeoxy ribonucleotide, or longer antisense RNA sequence.