Highly selective aptamer‐molecularly imprinted polymer hybrids for recognition of SARS‐CoV‐2 spike protein variants

Virus recognition has been driven to the forefront of molecular recognition research due to the COVID-19 pandemic. Development of highly sensitive recognition elements, both natural and synthetic is critical to facing such a global issue. However, as viruses mutate, it is possible for their recognition to wane through changes in the target substrate, which can lead to detection avoidance and increased false negatives. Likewise, the ability to detect specific variants is of great interest for clinical analysis of all viruses. Here, a hybrid aptamer-molecularly imprinted polymer (aptaMIP), that maintains selective recognition for the spike protein template across various mutations, while improving performance over individual aptamer or MIP components (which themselves demonstrate excellent performance). The aptaMIP exhibits an equilibrium dissociation constant of 1.61 nM toward its template which matches or exceeds published examples of imprinting of the spike protein. The work here demonstrates that “fixing” the aptamer within a polymeric scaffold increases its capability to selectivity recognize its original target and points toward a methodology that will allow variant selective molecular recognition with exceptional affinity

RNA Aptamers Generated against Oligomeric Aβ40 Recognize Common Amyloid Aptatopes with Low Specificity but High Sensitivity

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid β-protein (Aβ) oligomers. Aβ oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Aβ (Aβ40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Aβ40 oligomers but reacted with fibrils of Aβ40, Aβ42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Aβ40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with ≥15-fold higher sensitivity than thioflavin T (ThT), revealing substantial β-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect β-sheet formation, suggests that they can serve as superior amyloid recognition tools

Crossing borders to bind proteins—a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug desig

Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

New techniques for jet calibration with the ATLAS detector

A determination of the jet energy scale is presented using proton–proton collision data with a centre-of-mass energy of √s = 13 TeV, corresponding to an integrated luminosity of 140 fb−1 collected using the ATLAS detector at the LHC. Jets are reconstructed using the ATLAS particle-flow method that combines charged-particle tracks and topo-clusters formed from energy deposits in the calorimeter cells. The anti-kt jet algorithm with radius parameter R=0.4 is used to define the jet. Novel jet energy scale calibration strategies developed for the LHC Run 2 are reported that lay the foundation for the jet calibration in Run 3. Jets are calibrated with a series of simulation-based corrections, including state-of-the-art techniques in jet calibration such as machine learning methods and novel in situ calibrations to achieve better performance than the baseline calibration derived using up to 81 fb−1 of Run 2 data. The performance of these new techniques is then examined in the in situ measurements by exploiting the transverse momentum balance between a jet and a reference object. The b-quark jet energy scale using particle flow jets is measured for the first time with around 1% precision using γ+jet events

Zawartość amin biogennych w serze pleśniowym w trakcie przechowywania

The aim of this research was to study the formation of seven biogenic amines (histamine, agmatinc, spermine, spermidine, cadaverine, putrescine and tyramine) in three commercial mould cheeses from three different producers from the area of the Central Europę during 8-week storage in refrigerator at 6 š 2 °C. The analysis of biogenic amines was madr every week during 8-week of storage. Biogenic amines were extracted from the mould cheese by diluted HCl and determined using ion-exchange chromatography with post-column ninhydrin detection. Spermidine, spermine, putrescine and cadaverine were detected in tested mould cheeses. Spermidinc was quantitatively the most important biogenic arnuie in all samples. While spermidine was detected immediately after purchase of samples, the rest of detected biogenic amines were developed during storage. The amount of putrescine was mostly increased during storage while the concentration of spermidine was decreased during storage. However, after 8 weeks of storage all samples contained toxicologically insignificant concentrations of detected biogenic amines in comparison with EU legislation and scientific literature and can be considered to be safe for human health.Celem pracy było zbadanie syntezy siedmiu amin biogennych (histaminy, agmatyny, sperminy, spermidyny, kadaweryny, putrescyny i tyraminy) w trzech komercyjnie dostępnych serach pleśniowych pochodzących od różnych producentów z Europy Środkowej w czasie 8-tygodniowego przechowywania w lodówce w temperaturze 6 š 2°C. Oznaczenia poziomu amin biogennych wykonywano raz w tygodniu. Aminy biogenne były izolowane z sera pleśniowego przez rozcieńczony HC1 i oznaczane metodą chromatografii jonowymiennej i postkolumnowej reakcji ninhydrynowej. W badanych serach wykryto obecność spermidyny, sperminy, putrescyny i kadaweryny. W największych ilościach występowała spermidyna. Związek ten wykrywano w świeżo wyprodukowanym serze, natomiast pozostałe aminy pojawiały się stopniowo w czasie przechowywania. Największy wzrost stężenia w czasie przechowywania stwierdzono w przypadku putrescyny. Natomiast poziom spermidyny zmniejszał się w czasie przechowywania. Po 8 tygodniach przechowywania badane sery zawierały jednak nieznaczne ilości amin biogennych, w stężeniach dopuszczalnych przez normy UE i bezpiecznych dla ludzkiego zdrowia

A Compact Functional Quantum Dot-DNA Conjugate: Preparation, Hybridization and Specific Label-free DNA Detection

In this letter, we report the preparation of a compact, functional quantum dot (QD)-DNA conjugate, where the capturing target DNA is directly and covalently coupled to the QD surface. This enables the control of the separation distance between the QD donor and dye acceptor to within the range of the Förster radius. Moreover, a tri(ethylene glycol) linker is introduced to the QD surface coating to effectively eliminate the strong, non-specific adsorption of DNA on the QD surface. As a result, this QD-DNA conjugate hybridizes specifically to its complementary DNA with a hybridization rate constant comparable to that of free DNAs in solution. We show this system is capable of specific detection of nanomolar unlabeled complimentary DNA at low DNA probe:QD copy numbers via a ‘signal-on’ fluorescence resonance energy transfer (FRET) response