Forecasting the Reaction of DNA Modifying Enzymes on DNA Nanostructures by Coarse Grained Model for Stimuli-Responsive Drug Delivery

The reactivity of DNA modifying enzymes on their natural nucleic acid substrates has been fully understood. However, their reactivity on self-assembled nanostructures of nucleic acid is complicated and unpredictable. Here, we employed the molecular dynamic simulation to forecast the reactivity of tumor biomarker enzymes on DNA nanotubes by coarse grained model. It is found that the enzyme accessibility and the potential energy of the reaction products co-determine the structural change of DNA nanotubes. The reactivity can be regulated by the position of enzyme recognition site. According to the simulation results, stimuli-responsive drug nanocarrier with superior sensitivity and selectivity was developed. Drug payloads released in cancer cells is 3.7~5.5-fold higher than that in normal cells. The DNA nanocarrier equipped with cancer-specific aptamer AS1411 is used to deliver doxorubicin (DOX) to tumor-bearing mice not only effectively inhibiting tumor growth but also protecting major organs from drug-caused damage. This work provides new insights into the enzymatic reactivity of DNA nanostructures enriching the library of DNA-based reactions and heralding broad applications in nanomedicine.

Filling gaps of genome scaffolds via probabilistic searching optical maps against assembly graph

Background Optical maps record locations of specific enzyme recognition sites within long genome fragments. This long-distance information enables aligning genome assembly contigs onto optical maps and ordering contigs into scaffolds. The generated scaffolds, however, often contain a large amount of gaps. To fill these gaps, a feasible way is to search genome assembly graph for the best-matching contig paths that connect boundary contigs of gaps. The combination of searching and evaluation procedures might be “searching followed by evaluation”, which is infeasible for long gaps, or “searching by evaluation”, which heavily relies on heuristics and thus usually yields unreliable contig paths. Results We here report an accurate and efficient approach to filling gaps of genome scaffolds with aids of optical maps. Using simulated data from 12 species and real data from 3 species, we demonstrate the successful application of our approach in gap filling with improved accuracy and completeness of genome scaffolds. Conclusion Our approach applies a sequential Bayesian updating technique to measure the similarity between optical maps and candidate contig paths. Using this similarity to guide path searching, our approach achieves higher accuracy than the existing “searching by evaluation” strategy that relies on heuristics. Furthermore, unlike the “searching followed by evaluation” strategy enumerating all possible paths, our approach prunes the unlikely sub-paths and extends the highly-probable ones only, thus significantly increasing searching efficiency.

A Reinforcement Learning Framework for Pooled Oligonucleotide Design

The goal of oligonucleotide (oligo) design is to select oligos that optimize a set of design criteria. Oligo design problems are combinatorial in nature and require computationally intensive models to evaluate design criteria. Even relatively small problems can be intractable for brute-force approaches that test every possible combination of oligos, so heuristic approaches must be used to find near-optimal solutions. We present a general reinforcement learning framework, called OligoRL, to solve oligo design problems with complex constraints. OligoRL allows "black-box" design criteria and can be adapted to solve many oligo design problems. We highlight the flexibility of OligoRL by building tools to solve three distinct design problems: 1.) finding pools of random DNA barcodes that lack restriction enzyme recognition sequences (CutFreeRL); 2.) compressing large, non-degenerate oligo pools into smaller degenerate ones (OligoCompressor); and 3.) finding Not-So-Random hexamer primer pools that avoid rRNA and other unwanted transcripts during RNA-seq library preparation (NSR-RL). OligoRL demonstrates how reinforcement learning offers a general solution for complex oligo design problems. OligoRL and its associated software tools are available as a Julia package at http://jensenlab.net/tools.

Structural Landscape of the Transition from an ssDNA Dumbbell Plus Its Complementary Hairpin to a dsDNA Microcircle Via a Kissing Loop Intermediate

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii

Background Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity. Methods Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries. Results Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate. Conclusions We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase

A terminal uridylyl transferase site-specifically labels RNA with microenvironment-sensitive fluorescent nucleotides, which in turn provide direct read-outs to probe the enzyme recognition.

Mechanism of the secretion of the lanthipeptide nisin

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied example is nisin, which is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT which secrets nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the maturation machinery composed of the dehydratase NisB and the cyclase NisC. The data clearly demonstrated that the function of NisC, but the mere presence of NisB modulated the apparent secretion rates. Additional in vitro studies of detergent-solubilized NisT revealed how the activity of this ABC transporter is again influenced by the enzymes of the maturation machinery, but not the substrate.