Pairing nanoarchitectonics of oligodeoxyribonucleotides with complex diversity: concatemers and self-limited complexes.

The development of approaches to the design of two- and three-dimensional self-assembled DNA-based nanostructures with a controlled shape and size is an essential task for applied nanotechnology, therapy, biosensing, and bioimaging. We conducted a comprehensive study on the formation of various complexes from a pair of oligonucleotides with two transposed complementary blocks that can be linked through a nucleotide or non-nucleotide linker. A methodology is proposed to prove the formation of a self-limited complex and to determine its molecularity. It is based on the "opening" of a self-limited complex with an oligonucleotide that effectively binds to a duplex-forming block. The complexes assembled from a pair of oligonucleotides with different block length and different linker sizes and types were investigated by theoretical analysis, several experimental methods (a gel shift assay, atomic force microscopy, and ultraviolet melting analysis), and molecular dynamics simulations. The results showed a variety of complexes formed by only a pair of oligonucleotides. Self-limited associates, concatemer complexes, or mixtures thereof can arise if we change the length of a duplex and loop-forming blocks in oligonucleotides or via introduction of overhangs and chemical modifications. We postulated basic principles of rational design of native self-limited DNA complexes of desired structure, shape, and molecularity. Our foundation makes self-limited complexes useful tools for nanotechnology, biological studies, and therapeutics.

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens.

Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A-H1N1 and H3N2-and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0-M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

General Study and Gene Expression Profiling of Endotheliocytes Cultivated on Electrospun Materials.

Endothelization of the luminal surface of vascular grafts is required for their long-term functioning. Here, we have cultivated human endothelial cells (HUVEC) on different 3D matrices to assess cell proliferation, gene expression and select the best substrate for endothelization. 3D matrices were produced by electrospinning from solutions of poly(D,L-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), and blends of PCL with gelatin (Gl) in hexafluoroisopropanol. Structure and surface properties of 3D matrices were characterized by SEM, AFM, and sessile drop analysis. Cell adhesion, viability, and proliferation were studied by SEM, Alamar Blue staining, and 5-ethynyl-2'-deoxyuridine (EdU) assay. Gene expression profiling was done on an Illumina HiSeq 2500 platform. Obtained data indicated that 3D matrices produced from PCL with Gl and treated with glutaraldehyde provide the most suitable support for HUVEC adhesion and proliferation. Transcriptome sequencing has demonstrated a minimal difference of gene expression profile in HUVEC cultivated on the surface of these matrices as compared to tissue culture plastic, thus confirming these matrices as the best support for endothelization.

DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

Room-temperature distance measurements using RIDME and the orthogonal spin labels trityl/nitroxide.

Electron paramagnetic resonance (EPR) based nanometer distance measurements at ambient temperatures are of particular interest for structural biology applications. The nitroxide spin labels commonly used in EPR reveal relatively short transverse relaxation under these conditions, which limits their use for detecting static dipolar interactions. At the same time, the longitudinal relaxation of nitroxide spin labels is still long enough to allow using them as 'pumped' species in the relaxation induced dipolar modulation enhancement (RIDME) experiment where the detection is carried out on the slower relaxing triarylmethyl (TAM) spin labels. In the present study, we report the first demonstration of room-temperature RIDME distance measurements in nucleic acids using TAM as the slow-relaxing detected species and traditional nitroxide as the fast-relaxing partner spin. Two types of immobilizers, glassy trehalose and the modified silica gel Nucleosil, were used for immobilization of the spin-labeled biomolecules. The room-temperature RIDME-based distance distributions are in good agreement with those measured at 80 K by other techniques. Room-temperature RIDME on the spin pairs trityl/nitroxide may become a useful method for the structural characterization of biomacromolecules and biomolecular complexes at near physiological temperatures.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies.

A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures.

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

Dynamical transition in molecular glasses and proteins observed by spin relaxation of nitroxide spin probes and labels.

In glassy substances and biological media, dynamical transitions are observed in neutron scattering that manifests itself as deviations of the translational mean-squared displacement, 〈x2〉, of hydrogen atoms from harmonic dynamics. In biological media, the deviation occurs at two temperature intervals, at ∼100-150 K and at ∼170-230 K, and it is attributed to the motion of methyl groups in the former case and to the transition from harmonic to anharmonic or diffusive motions in the latter case. In this work, electron spin echo (ESE) spectroscopy-a pulsed version of electron paramagnetic resonance-is applied to study the spin relaxation of nitroxide spin probes and labels introduced in molecular glass former o-terphenyl and in protein lysozyme. The anisotropic contribution to the rate of the two-pulse ESE decay, ΔW, is induced by spin relaxation appearing because of restricted orientational stochastic molecular motion; it is proportional to 〈α2〉τc, where 〈α2〉 is the mean-squared angle of reorientation of the nitroxide molecule around the equilibrium position and τc is the correlation time of reorientation. The ESE time window allows us to study motions with τc < 10-7 s. For glassy o-terphenyl, the 〈α2〉τc temperature dependence shows a transition near 240 K, which is in agreement with the literature data on 〈x2〉. For spin probes of essentially different size, the obtained data were found to be close, which evidences that motion is cooperative, involving a nanocluster of several neighboring molecules. For the dry lysozyme, the 〈α2〉τc values below 260 K were found to linearly depend on the temperature in the same way as it was observed in neutron scattering for 〈x2〉. As spin relaxation is influenced only by stochastic motion, the harmonic motions seen in ESE must be overdamped. In the hydrated lysozyme, ESE data show transitions near 130 K for all nitroxides, near 160 K for the probe located in the hydration layer, and near 180 K for the label in the protein interior. For this system, the two latter transitions are not observed in neutron scattering. The ESE-detected transitions are suggested to be related with water dynamics in the nearest hydration shell: with water glass transition near 130 K and with the onset of overall water molecular reorientations near 180 K; the disagreement with neutron scattering is ascribed to the larger time window for ESE-detected motions.

Interaction of triarylmethyl radicals with DNA termini revealed by orientation-selective W-band double electron-electron resonance spectroscopy.

Spin labels selectively attached to biomolecules allow high-accuracy nanoscale distance measurements using pulsed electron paramagnetic resonance (EPR), in many cases providing the only access to the structure of complex biosystems. Triarylmethyl (TAM) radicals have recently emerged as a new class of spin labels expanding the applicability of the method to physiological temperatures. Along with other factors, the accuracy of the obtained distances crucially relies on the understanding of interactions between biomolecules and spin labels. In this work, we consider such crucial interactions and their impact on pulsed EPR distance measurements in TAM-labeled DNAs. Using orientation-selective high-frequency (94 GHz) double electron-electron resonance (DEER) we demonstrate strong specific interactions between DNA termini and TAM labels, leading to a significant restriction of their conformational mobility. An understanding of such interactions guides the way to select optimum TAM-labeling strategies, thus refining nanoscale EPR distance measurements in nucleic acids and their complexes under physiological conditions.

Saccharides as Prospective Immobilizers of Nucleic Acids for Room-Temperature Structural EPR Studies.

Pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for structural studies of biomolecules and their complexes. This method, whose applicability has been recently extended to room temperatures, requires immobilization of the studied biosystem to prevent averaging of dipolar couplings; at the same time, the modification of native conformations by immobilization must be avoided. In this work, we provide first demonstration of room-temperature EPR distance measurements in nucleic acids using saccharides trehalose, sucrose, and glucose as immobilizing media. We propose an approach that keeps structural conformation and unity of immobilized double-stranded DNA. Remarkably, room-temperature electron spin dephasing time of triarylmethyl-labeled DNA in trehalose is noticeably longer compared to previously used immobilizers, thus providing a broader range of available distances. Therefore, saccharides, and especially trehalose, can be efficiently used as immobilizers of nucleic acids, mimicking native conditions and allowing wide range of structural EPR studies at room temperatures.

Study of a DNA Duplex by Nuclear Magnetic Resonance and Molecular Dynamics Simulations. Validation of Pulsed Dipolar Electron Paramagnetic Resonance Distance Measurements Using Triarylmethyl-Based Spin Labels.

Pulse dipole-dipole electron paramagnetic resonance (EPR) spectroscopy (double electron-electron resonance [DEER] or pulse electron-electron double resonance [PELDOR] and double quantum coherence [DQC]) allows for measurement of distances in biomolecules and can be used at low temperatures in a frozen solution. Recently, the possibility of distance measurement in a nucleic acid at a physiological temperature using pulse EPR was demonstrated. In these experiments, triarylmethyl (TAM) radicals with long memory time of the electron spin served as a spin label. In addition, the duplex was immobilized on modified silica gel particles (Nucleosil DMA); this approach enables measurement of interspin distances close to 4.5 nm. Nevertheless, the possible influence of TAM on the structure of a biopolymer under study and validity of the data obtained by DQC are debated. In this paper, a combination of molecular dynamics (MD) and nuclear magnetic resonance (NMR) methods was used for verification of interspin distances measured by the X-band DQC method. NMR is widely used for structural analysis of biomolecules under natural conditions (room temperature and an aqueous solution). The ultraviolet (UV) melting method and thermal series (1)H NMR in the range 5-95 °C revealed the presence of only the DNA duplex in solution at oligonucleotide concentrations 1 µM to 1.1 mM at temperatures below 40 °C. The duplex structures and conformation flexibility of native and TAM-labeled DNA complexes obtained by MD simulation were the same as the structure obtained by NMR refinement. Thus, we showed that distance measurements at physiological temperatures by the X-band DQC method allow researchers to obtain valid structural information on an unperturbed DNA duplex using terminal TAM spin labels.

Room-temperature electron spin relaxation of nitroxides immobilized in trehalose: Effect of substituents adjacent to NO-group.

Trehalose has been recently promoted as efficient immobilizer of biomolecules for room-temperature EPR studies, including distance measurements between attached nitroxide spin labels. Generally, the structure of nitroxide influences the electron spin relaxation times, being crucial parameters for room-temperature pulse EPR measurements. Therefore, in this work we investigated a series of nitroxides with different substituents adjacent to NO-moiety including spirocyclohexane, spirocyclopentane, tetraethyl and tetramethyl groups. Electron spin relaxation times (T1, Tm) of these radicals immobilized in trehalose were measured at room temperature at X- and Q-bands (9/34GHz). In addition, a comparison was made with the corresponding relaxation times in nitroxide-labeled DNA immobilized in trehalose. In all cases phase memory times Tm were close to 700ns and did not essentially depend on structure of substituents. Comparison of temperature dependences of Tm at T=80-300K shows that the benefit of spirocyclohexane substituents well-known at medium temperatures (∼100-180K) becomes negligible at 300K. Therefore, unless there are specific interactions between spin labels and biomolecules, the room-temperature value of Tm in trehalose is weakly dependent on the structure of substituents adjacent to NO-moiety of nitroxide. The issues of specific interactions and stability of nitroxide labels in biological media might be more important for room temperature pulsed dipolar EPR than differences in intrinsic spin relaxation of radicals.

Nanorings from Concatemeric DNA: Chemical Modification Drives Nanostructure Formation.

Self-assembly of DNA concatemers from native duplexes and those containing non-nucleotidic bridges of varying polarity composed of repeating oligo(ethylene glycol) phosphates -O(CH2CH2O)(n)PO2- or α,Ω-alkanediol phosphates -O(CH2)10OPO2(-)- units was compared. The structures obtained were characterised by polyacrylamide gel electrophoresis, enzymatic digestion and AFM. Our results have revealed that chemically-modified duplexes favour self-termination of concatemer growth and yield up to 35% of nanosized DNA rings.

Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs.

Triarylmethyl (trityl, TAM) based spin labels represent a promising alternative to nitroxides for EPR distance measurements in biomolecules. Herewith, we report synthesis and comparative study of series of model DNA duplexes, 5'-spin-labeled with TAMs and nitroxides. We have found that the accuracy (width) of distance distributions obtained by double electron-electron resonance (DEER/PELDOR) strongly depends on the type of radical. Replacement of both nitroxides by TAMs in the same spin-labeled duplex allows narrowing of the distance distributions by a factor of 3. Replacement of one nitroxide by TAM (orthogonal labeling) leads to a less pronounced narrowing but at the same time gains sensitivity in DEER experiment due to efficient pumping on the narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide pairs are influenced by the structure of the linker: the use of a short amine-based linker improves the accuracy by a factor of 2. At the same time, a negligible dependence on the linker length is found for the distribution width in TAM/TAM pairs. Molecular dynamics calculations indicate greater conformational disorder of nitroxide labels compared to TAM ones, thus rationalizing the experimentally observed trends. Thereby, we conclude that double spin-labeling using TAMs allows obtaining narrower spin-spin distance distributions and potentially more precise distances between labeling sites compared to traditional nitroxides.

Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy.

Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).