A Simple Microfluidic Assay for the Detection of Ligation Product

2015 ◽  
Vol 19 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Lei Zhang ◽  
Jingjing Wang ◽  
Johann Roebelen ◽  
Anubhav Tripathi
Keyword(s):  
Synthesis ◽  
2019 ◽  
Vol 51 (05) ◽  
pp. 1273-1283 ◽  
Author(s):  
Simon Baldauf ◽  
Jeffrey Bode

The α-ketoacid–hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. The most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. While very effective, monomers that give canonical amino acid residues are in high demand. In order to preserve the stability and reactivity of cyclic hydroxylamines, but form a canonical amino acid residue upon ligation, we sought to prepare cyclic derivatives of serine hydroxylamine. An evaluation of several cyclization strategies led to cyclobutanone ketals as the leading structures. The preparation, stability, and amide-forming ligation of these serine-derived ketals are described.


2018 ◽  
Vol 2018 (8) ◽  
pp. pdb.prot097865 ◽  
Author(s):  
Tae Hoon Kim ◽  
Job Dekker
Keyword(s):  

Author(s):  
Ping Xu ◽  
Martina Billmeier ◽  
Irina Mohorianu ◽  
Darrell Green ◽  
William D Fraser ◽  
...  

AbstractNext generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with greatly reduced proportion of adapter-adapter products due to the degradation of nonligated 3’ adapters. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue.


2018 ◽  
Vol 115 (15) ◽  
pp. 3752-3757 ◽  
Author(s):  
Daniel L. Dunkelmann ◽  
Yuki Hirata ◽  
Kyle A. Totaro ◽  
Daniel T. Cohen ◽  
Chi Zhang ◽  
...  

The facile rearrangement of “S-acyl isopeptides” to native peptide bonds via S,N-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, “O-acyl isopeptides” derived from hydroxy-asparagine [aspartic acid-β-hydroxamic acid; Asp(β-HA)] rearrange to form native peptide bonds via an O,N-acyl shift. This process constitutes a rare example of an O,N-acyl shift that proceeds rapidly across a medium-size ring (t1/2 ∼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form O-acyl isopeptides only by the use of highly activated acyl donors and appropriate protecting groups in organic solvent, Asp(β-HA) is sufficiently reactive to form O-acyl isopeptides by treatment with an unprotected peptide-αthioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected N-terminal Asp(β-HA)-peptide and peptide-αthioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.


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