Diversity-Oriented Enzymatic Modular Assembly of ABO Histo-blood Group Antigens

Enzymatic synthesis of all 15 naturally occurring human ABH antigens was achieved using a diversity-oriented enzymatic modular assembly (EMA) strategy. Three enzyme modules were developed, each one-pot multienzyme module comprises a glycosyltransferase and one or two corresponding sugar nucleotide generating enzyme(s). These multienzyme cascade processes provide an efficient and convenient platform for collective synthesis of all 15 ABH antigens in three operationally simple steps from five readily available disaccharide acceptors and three simple free sugars as donor precursors

Chemoenzymatic synthesis of tumor-associated antigen N3 minor octasaccharide

<p></p> <p>A highly efficient chemoenzymatic approach was developed for the preparative scale synthesis of complex tumor-associated antigen N3 minor octasaccharide. The success of this approach relies on the highly efficient one-pot multi-enzyme based sequential enzymatic elongation of a chemically synthesized branched tetrasaccharide core structure.</p

Regioselective Chemoenzymatic Synthesis of Ganglioside Disialyl Tetrasaccharide Epitopes

A novel chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes found as the terminal oligosaccharides of GD1α, GT1aα, and GQ1bα is described. It relies on chemical manipulation of enzymatically generated trisaccharides as conformationally constrained acceptors for regioselective enzymatic α2–6-sialylation. This strategy provides a new route for easy access to disialyl tetrasaccharide epitopes and their derivatives

Regioselective One-Pot Benzoylation of Triol and Tetraol Arrays in Carbohydrates

Protection of 2,3,4-<i>O</i>-unprotected α-galacto- and α-fucopyranosides with BzCN and DMAP/DIPEA as the base afforded directly and regioselectively the 3-<i>O</i>-unprotected derivatives. The rationale for these studies was to take advantage of the eventual cooperativity of the “cyanide effect” and “the alkoxy group mediated diol effect”. This way, even the totally unprotected α-galactopyranosides could be regioselectively transformed into the corresponding 2,4,6-<i>O</i>-protected derivatives. The great utility of these building blocks was demonstrated in efficient trisaccharide syntheses

Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation

A β-galactoside α2,6-sialyltransferase from <i>Photobacterium damselae</i> (Pd2,6ST) that is capable of sialylating both terminal and internal galactose and <i>N</i>-acetylgalactosamine was herein redesigned for regioselectively producing terminal α2,6-sialosides. Guided by a recently developed bump-hole strategy, a series of mutations at Ala200 and Ser232 sites were created for reshaping the acceptor binding pocket. Finally, a Pd2,6ST double mutant A200Y/S232Y with an altered L-shaped acceptor binding pocket was identified to be a superior α2,6-sialyltransferase which can efficiently catalyze the regioselective α2,6-sialylation of galactose or <i>N</i>-acetylgalactosamine at the nonreducing end of a series of glycans. Meanwhile, A200Y/S232Y remains flexible donor substrate specificity and is able to transfer Neu5Ac, Neu5Gc, and KDN

Insert-size distributions of long-range PE sequencing libraries.

<p>(A), 2- to 35-kb libraries; (B), 10 kb-WGA and 10 kb-dam libraries. The read-pairs that were uniquely mapped to the human genome (NCBI build 37) were used for this analysis. The insert size of a library and its corresponding small insert read contamination are shown in the ‘−’ and ‘+’direction of the x-axis, respectively. The ‘−’ direction represents the orientation relationship between PEs from circularized long-range DNA molecules (>1 kb) when mapped to the human genome, while ‘+’ represents that between the two ends from linear small DNA fragments (∼500 bp).</p

Two long insertions in YH genome detected by long-range PE.

<p>Mapping the long-range PE reads back to the human genome (NCBI build 37) resulted in the detection of a previously identified ∼8 kb insertion in chromosome 7 (A) and a novel ∼7 kb insertion in chromosome 14 (B) in the YH genome. The abnormally mapped PE reads that supported the insertions by showing unexpected short insert size are shown.</p

Comparison of long-range PE sequencing methods.

<p>(A–D) Long-range PE sequencing with linker oligonucleotides. In these methods, biotin-labeled linker oligonucleotides are added to the two ends of long-range DNA fragments, followed by enzymes-induced intra-molecule circularization, and recovery of the paired-end for sequencing. The addition of linker oligonucleotides and subsequent complex enzyme reactions require 5–8 recoveries before capturing the paired-ends from circularized DNA fragments. In addition, the use of expensive enzymes involves additional costs. (E), Long-range PE sequencing by direct intra-molecule ligation or molecular linker-free circularization. In the method, the 3′ends of long-range DNA fragments were biotin-labeled, followed by direct intra-molecule circularization and recovery of PE ends. This method requires less recovery steps (3–4) and no complex enzyme reaction system. The steps for DNA recovery are in bold. We applied the method E in this research.</p

Paired-End Sequencing of Long-Range DNA Fragments for <em>De Novo</em> Assembly of Large, Complex Mammalian Genomes by Direct Intra-Molecule Ligation

<div><h3>Background</h3><p>The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for <em>de novo</em> assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS.</p> <h3>Results</h3><p>We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data.</p> <h3>Conclusions</h3><p>In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the <em>de novo</em> assembly of a large, complex genome using NGS short reads.</p> </div

Summary of <i>de novo</i> YH genome assembly.

<p>The data from the YH project was used for the contig and initial scaffold assembly. Then, the long-range PE data were added step by step for scaffold construction. Genome coverage and gene coverage was calculated using the NCBI build 37 and RefSeq gene set as reference, respectively. The X and Y chromosomes were excluded while calculating genome coverage and gene coverage. For calculation of scaffold N50, N90 and total length, the intra-scaffold gaps were included.</p