Project description:The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2-4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.
Project description:Proof-of-concept of a new method involving the limited digestion and subsequent ligation of intramolecular RNA structures in situ followed by deep sequencing Proof-of-concept of RPL in S. cerevisiae and H. sapiens tissue culture
Project description:Proof-of-concept of a new method involving the limited digestion and subsequent ligation of intramolecular RNA structures in situ followed by deep sequencing
Project description:We revealed a large population of long cell-free DNA molecules (up to 23,635 bp in length) in maternal plasma and developed an approach which leveraged the abundance of CpG sites on long molecules to deduce the tissue of origin of individual plasma DNA molecules based on single-molecule methylation analysis. We illustrated how such an approach may be utilized to achieve noninvasive prenatal testing of monogenic diseases. We also revealed a reduction in amounts of such long cell-free DNA molecules and a different end motif profile in maternal plasma DNA from pregnancies with preeclampsia.
Project description:Changes in the amino acid sequences of proteins cause thousands of human genetic diseases. However, only a subset of variants in any protein is typically pathogenic, with variants having a diversity of molecular consequences. Determining which of the thousands of possible variants in any protein have similar molecular effects is very challenging, but crucial for identifying pathogenic variants, determining disease mechanisms, understanding clinical phenotypic variation, and developing targeted therapeutics. Here we present a general method to classify variants by their molecular effects that we term intramolecular genetic interaction profiling. The approach relies on the principle that variants with similar molecular consequences have similar genetic interactions with other variants in the same protein. These intramolecular genetic interactions are straightforward to quantify for any protein with a selectable function. We apply intramolecular genetic interaction profiling to amyloid beta, the protein that aggregates in Alzheimer’s disease (AD) and is mutated in familial AD (fAD). Genetic interactions identify two classes of gain-of-function variants, with all known familial Alzheimer’s disease variants having very similar genetic interaction profiles, consistent with a common gain-of-function mechanism leading to pathology. We believe that intramolecular genetic interaction profiling is a powerful approach for classifying variants in disease genes that will empower rare variant association studies and the discovery of disease mechanisms.
Project description:Efforts to identify ccRNA by sequencing by gibson circularization after template-switching 5' RACE using a cRNA-end specific primer and subsequent sequencing by Illumina miSeq. Sequencing of wild-type A/WSN/1933 and a variant bearing the mutation T677A in the PB1 subunit.