Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence

Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and 5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.     

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.     

Complex protein patterns formation via salt-induced self-assembly and droplet evaporation

Complex and elegant protein patterns in rosette, scallop, Chinese arrow and dendrite shapes at macroscopic length scales were prepared using salt-induced molecular self-assembly and droplet evaporation methods. The direct visual observation method using fluorescence microscopy was adopted to characterize the formation of these protein patterns in situ. Further studies from an optical interferometric profiler have shown that both rosette and scalloped protein patterns are hierarchical structures of concentric rings consisting of many prism-like columnar stacks, with each of the stack having thousands of protein molecules. Systematic experimental studies were performed to investigate the influence of salt concentration, protein concentration and evaporation rate on the morphologies of protein patterns. Upon the analysis of the representative fluorescent microscope images some theoretical explanations, based on Deegan’s theory on the “coffee ring” effect and the dynamic self-assembly mechanism, were proposed to illustrate the dynamics for the formation of different protein patterns. Two different evaporation modes have been found: edge-enhanced evaporation for low salt concentration solutions, i.e., the higher evaporation rate exists at the edge of the droplet; center-enhanced evaporation for high salt concentration solutions, in which faster evaporation occurs at the droplet center consisting of a lot of crystallized salts.     

InspIRED by Nature: NADPH‐Dependent Imine Reductases (IREDs) as Catalysts for the Preparation of Chiral Amines

Imine reductases (IREDs) are NADPH‐dependent oxidoreductases that catalyse the asymmetric reduction of cyclic prochiral imines to amines, with excellent stereoselectivity. Since their discovery, stereocomplementary IREDs have been applied to the production of both (S) and (R) cyclic secondary amines, and the expansion in gene sequences recently identified has hinted at new substrate ranges that extend into acyclic imines and even suggest the possibility of asymmetric reductive amination from suitable ketone and amine precursors. Structural studies of various IREDs are beginning to reveal the complexities inherent in determining substrate range, stereoselectivity and mechanism in these enzymes, which represent a valuable emerging addition to the toolbox of available biocatalysts for chiral amine production.     

Pattern formation in a cavity longer than the coherence length of the light in it

We study, theoretically and experimentally, the evolution of patterns in a passive nonlinear cavity that is longer than the coherence length of the light circulating in it. The patterns exhibit spatial line narrowing as the feedback is increased, resembling the line narrowing in lasers.     

Collisions between optical spatial solitons propagating in opposite directions

We formulate the theory describing the evolution and interactions between optical spatial solitons that propagate in opposite directions. We show that coherent collisions between counterpropagating solitons give rise to a new focusing mechanism resulting from the interference between the beams, and that interactions between such solitons are insensitive to the relative phase between the beams.