No abstract

Surface-enhanced Raman spectroscopy (SERS) is considered a highly promising technology for different analytical purposes. The applications of SERS are still quite limited due its relatively poor quantitative repeatability and the fact that SERS is very sensitive to oxidation, which is a challenge especially with silver based SERS substrates. Here, the link between these phenomena is investigated by exposing silver SERS substrates to ambient laboratory air. We show that SERS intensity decreases exponentially after the exposure, which consequently leads to an increasing standard deviation (σ) in intensity. Within a five-hour measurement window, the SERS intensity already drops by 60%, while σ triples from 7% to 21%. The SERS results are supplemented by elemental analysis, which shows that oxidation and atmospheric carbon contamination coincide with the rapid SERS intensity decrease. The results emphasize how sensitive SERS is towards atmospheric contamination and how it can also reduce the measurement repeatability – even if the substrates are exposed to air just for a very short period of time.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

Surface enhanced Raman scattering (SERS) fiber probes have enormous potential in optical sensing applications. However, their widespread use has been hindered by two major obstacles: the difficulty of fabricating the required silver nanostructures on optical fibers and the tarnishing of silver, rapidly degrading their sensing properties. Here we propose a solution to these dilemmas by abandoning the use of metallic silver and conventional nanofabrication procedures. Instead, we base our fabrication on chemically stable silver chloride and show that it can be directly grown on the optical fibers without any advanced fabrication equipment. As silver chloride itself is not SERS-active, we demonstrate how to “activate” the probes by turning the crystals into metallic silver nanostructures via photoreduction. We verify that if stored in the non-activated stage, the sensing properties of the structures remain unchanged. Finally, we demonstrate the high sensitivity (signal-to-noise ratio up to 42 ± 3 dB) of the probes in real-time in situ measurements at nanomolar analyte concentrations.

DNA polymerases α, δ and ε are large multisubunit complexes that replicate the bulk of the DNA in the eukaryotic cell. In addition to the homologous catalytic subunits, these enzymes possess structurally related B subunits, characterized by a carboxyterminal calcineurin-like and an aminoproximal oligonucleotide/oligosaccharide binding-fold domain. The B subunits also share homology with the exonuclease subunit of archaeal DNA polymerases D. Here, we describe a novel domain specific to the N-terminus of the B subunit of eukaryotic DNA polymerases ε. The N-terminal domain of human DNA polymerases ε (Dpoe2NT) expressed in Escherichia coli was characterized. Circular dichroism studies demonstrated that Dpoe2NT forms a stable, predominantly α-helical structure. The solution structure of Dpoe2NT revealed a domain that consists of a left-handed superhelical bundle. Four helices are arranged in two hairpins and the connecting loops contain short β-strand segments that form a short parallel sheet. DALI searches demonstrated a striking structural similarity of the Dpoe2NT with the α-helical subdomains of ATPase associated with various cellular activity (AAA+) proteins (the C-domain). Like C-domains, Dpoe2NT is rich in charged amino acids. The biased distribution of the charged residues is reflected by a polarization and a considerable dipole moment across the Dpoe2NT. Dpoe2NT represents the first C-domain fold not associated with an AAA+ protein.