Simple and Practical DNA Quantification Method for DNA-Encoded Library Synthesis.

Over the past three decades, DNA-encoded library (DEL) technologies have become one of the most relevant strategies for hit-finding. Recent advances in synthetic methodologies for DNA-encoded libraries rendered the increased chemical space available, but it is unknown how every variety of chemistry affects DNA's integrity. Available assays to quantify DNA damage are restricted to electrophoresis, ligation efficiency, and mostly qPCR quantification and sequencing, which may contain predisposition and inconsistency. We developed an external standard method through LC-MS analysis to accurately quantify DNA damage throughout the chemical transformations. An assessment was conducted on on-DNA chemical reactions that are frequently employed in DEL synthesis, and these results were compared to traditional qPCR measurements. Our study provides a simple, practicable, and accurate measurement for DNA degradation during DEL synthesis. Our finding reveals substantial disagreement among the usual DNA-damaging assessment methods, which have been largely neglected so far.

Constructive on-DNA Thiol Aerial Oxidization for DNA-Encoded Library Synthesis.

A novel on-DNA oxidative disulfide formation method has been developed. Under ambient conditions, the methodology showcased wide applicability and swift implementation in routine DNA-encoded library synthesis to access pharmaceutically relevant motifs.

Development of the Inverse Sonogashira Reaction for DEL Synthesis.

An efficient approach for aryl acetylene DNA-encoded library (DEL) synthesis was developed in this study by transition-metal-mediated inverse Sonogashira reaction of 1-iodoalkyne with boronic acid under ambient conditions, with moderate to excellent conversions and broad substrate adaptability for the first time. Compared to palladium-phosphine, copper iodide performed better in the on-DNA inverse Sonogashira reaction. Interestingly, substrate diversity can be enhanced by first interrogating coupling reagents under copper-promoted conditions, and then revalidating them under palladium-facilitated conditions for those reagents which failed under the former. This complementary validation strategy is particularly well-fitted to any DEL validation studies.

Expression and function analysis of crustacyanin gene family involved in resistance to heavy metal stress and body color formation in Exopalaemon carinicauda.

Crustacyanin has the function of binding astaxanthin which is the best antioxidant, and plays an important role in the body color variation of crustaceans. To investigate the causes of body color variation of the ridgetail white prawn, Exopalaemon carinicauda, the present study obtained four subtypes of crustacyanin gene: C1, C2, A1, and A2. Based on fluorescence quantitative polymerase chain reaction, lipocalin-C1 is mainly expressed in the eyestalk, lipocalin-C2 is in the ventral nerve cord, and lipocalin-A1 and lipocalin-A2 are in subcutaneous adipose tissues. Under the inhibiting effect of Cd2+ stress, the expression of four subtypes first increases and then decreases within 24 h, and reaches the maximum at 6 or 12 h. RNA interference experiments showed a decrease in the expression of lipocalin genes in subcutaneous adipose tissue for each subtype, with the body color changing from transparent to red, and the dark red spots on the epidermis changing to bright red. Moreover, the blue protein in the subcutaneous adipose tissue largely disappeared, based on the light micrographs. In view of these findings, the crustacyanin gene appears to fulfill some function in the resistance to heavy metal stress and body color formation of E. carinicauda.

Cloning and functional study of lipocalin: retinol-binding protein-like gene family of the ridgetail white prawn, Exopalaemon carinicauda.

Lipocalin is a large family with complex functions including retinol-binding protein (RBP), crustacyanin (CRCN), apolipoprotein D, etc. In shrimps, it is well known that CRCN is related to body color. Recently, retinoic acid/retinol-binding protein was found in shrimp. However, little is known about the function of RBP and relationships among the gene members of lipocalin in shrimps. Based on the transcriptome sequences responding to starvation stress, three genes of the lipocalin-retinol-binding protein-like gene family (lipocalin-1, lipocalin-2, and lipocalin-3) were cloned by RACE from the ridgetail white prawn, Exopalaemon carinicauda. Homology analysis showed that these three genes had high similarity with the known insect apolipoprotein D gene and vertebrate retinol-binding protein gene, and they are of the same type in terms of evolution. Fluorescence quantitative PCR showed that the above three genes were mainly expressed in the ventral nerve cord of E. carinicauda. The expression characteristics of the three genes at different developmental stages showed that they were more highly expressed at the larval stage, which suggests that they might be related to embryonic and larval development. The RNA interference tests showed that after silencing lipocalin-1 and lipocalin-3, the body color of individual shrimps turned slightly red and the blue pigment in the epidermis largely disappeared, but no significant change took place in the appearance of individuals after silencing lipocalin-2. In addition, on the 6th and 16th days of interference, dead shrimps appeared in the lipocalin-1 and lipocalin-3 interference groups. The dead shrimps had hard crusts and remained in a molting posture. Totally, this study showed that the retinol-binding protein-like gene obtained in this study had certain biological functions in the growth and development and body color formation as CRCN; in addition, it also plays a role in nerve system and molting of E. carinicauda.