Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote “micrometer-sized DNA track” (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3′ end and ≈1000 repeating sequence units at its 5′ end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes.     

Hybridization Detection of Osmium Tetroxide Bipyridine‐Labeled DNA and RNA on Heated Gold Wire Electrodes

We report about hybridization detection of different nucleic acids on capture probe‐modified heated gold wire electrodes. We have compared three kinds of nucleic acid targets: DNA, uracil‐conjugated DNA, and RNA. All three sorts of nucleic acids targets could be labeled with osmium tetroxide bipyridine, hybridized with immobilized DNA capture probes and then detected by square‐wave voltammetry. Heating the gold electrode instead of the entire bulk hybridization solution leads to improved hybridization efficiency in most cases. The reason could be found in a thermal micro‐stirring effect around the heated wire electrode. Also selectivity was improved. Mismatches could be discriminated for DNA and uracil‐conjugated DNA targets. Mismatches in RNA strands, however, are more difficult to detect due to relatively stable secondary structures.     

ApA Cleavage Promoted by Oxa-aza Macrocycles and Their Zn(II) Complexes. The Role of pH and Metal Coordination in the Hydrolytic Mechanism

Abstract

The thermodynamic parameters for protonation and Zn(II) complex formation with ligand 1,4,7,16,19,22-hexaza-10,13,25,28-tetraoxacyclotriacontane (L1) have been determined. L1 forms stable dizinc complexes from neutral to alkaline pH. The hydrolytic ability toward adenylyl(3′-5′)adenosine (ApA) of L1 and its dizinc(II) complexes have been analyzed by means of HPLC chromatography. Only partially protonated species of L promote ApA hydrolysis suggesting that the cleavage process is cooperatively promoted by a general base catalysis by neutral amine groups and a general acid catalysis by protonated ammonium functions. Concerning the Zn(II) complexes, the hydrolysis rates increase in the presence of the hydroxo complexes [Zn₂L1(OH)]³⁺ and [Zn₂L1(OH)²]²⁺. This indicates that Zn-OH functions play a crucial role in the hydrolytic process, assisting the deprotonation of the 2′-OH group of ApA, which may act as nucleophile in the cleavage process. Both binuclear L1 complexes are better catalysts than the mononuclear [ZnL₂(OH)]⁺ complex (L₂ = 1,4-Dioxa-7,10,13-triazacyclopentadecane), indicating a cooperative role of the two Zn(II) ions in ApA cleavage by [Zn₂L1(OH)]³⁺ and [Zn₂L1(OH)₂]²⁺, probably due to a bridging coordination of the phosphate moiety of ApA to the two metal centers.     

A Polymer Reaction Engineering Approach to Polynucleotide Replication

It is often interesting to look at specific scientific questions from the perspective of other, related technologies. The problem of nonenzymatic RNA replication can be viewed from the perspective of classical polymer science and with some interesting insights. This communication will show that current mechanism of stalling, which is used to account for the fact that the rate of insertion errors in nonenzymatic RNA replication are much lower than would be expected, can be modeled by using the Mayo–Lewis equation for calculating copolymer composition in copolymerizations of synthetic polymers. Using kinetic parameters from the literature, it is possible to independently predict the error threshold for RNA replication. Agreement with the accepted model for nonenzymatic replication is surprisingly good.

     

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.     

Ground state and glass transition of the RNA secondary structure

RNA molecules form a sequence-specific self-pairing pattern at low temperatures. We analyze this problem using a random pairing energy model as well as a random sequence model that includes a base stacking energy in favor of helix propagation. The free energy cost for separating a chain into two equal halves offers a quantitative measure of sequence specific pairing. In the low temperature glass phase, this quantity grows quadratically with the logarithm of the chain length, but it switches to a linear behavior of entropic origin in the high temperature molten phase. Transition between the two phases is continuous, with characteristics that resemble those of a disordered elastic manifold in two dimensions. For designed sequences, however, a power-law distribution of pairing energies on a coarse-grained level may be more appropriate. Extreme value statistics arguments then predict a power-law growth of the free energy cost to break a chain, in agreement with numerical simulations. Interestingly, the distribution of pairing distances in the ground state secondary structure follows a remarkable power-law with an exponent -4/3, independent of the specific assumptions for the base pairing energies.     

复合物Ca+-RNA碱基的结构与稳定性

用密度泛函理论(DFT)中的杂化密度泛函B3LYP方法,在6311+G(2df,2p)基组水平上,确定了RNA碱基与Ca+的气相金属离子亲和能和复合物的优化结构.通过考虑Ca+作用于RNA碱基互变异构体的不同位置,获得了所有稳定的复合物.对于胞核嘧啶,最稳定的复合物起源于最稳定的分子互变异构体;对于胸腺嘧啶,最稳定的分子互变异构体形成的复合物的键能最小,而最不稳定的互变异构体形成的复合物键能最强;尿嘧啶的情形与胸腺嘧啶相似.在互变异构体复合物中,键能是依赖于金属离子成键的位置,而总能量取决于全部的原子和它们之间的相对位置,这可能是造成几种互变异构体复合物键能和总能量变化趋势并不一致的原因.     

Total synthesis of dehydroaltenusin

The first total synthesis of dehydroaltenusin, a natural enzyme inhibitor, is described. The key step involves Suzuki-coupling reaction of an aryl triflate prepared from 2,4,6-trihydroxybenzoic acid with a catechol-derived boronic acid or boronic ester. The synthetic product was evaluated as a potent inhibitor against eukaryotic DNA polymerase α and other DNA polymerases.