ssDNA Oligomers Research Articles

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

Mapping the Morphology of DNA on Carbon Nanotubes in Solution Using X-ray Scattering Interferometry.

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with potential applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes such as (GT)n-SWCNTs recognizing the neuromodulator, dopamine. It remains unclear how the ssDNA conformation on the SWCNT surface contributes to functionality, as observations have been limited to computational models or experiments under dehydrated conditions that differ substantially from the aqueous biological environments in which the nanosensors are applied. We demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two (GT)n ssDNA oligomer lengths (n = 6, 15) that are used on SWCNTs in the context of dopamine sensing and measure the ssDNA conformational changes as a function of ionic strength and during dopamine interaction. We show that the shorter oligomer, (GT)6, adopts a more periodically ordered ring structure along the SWCNT axis (inter-ssDNA distance of 8.6 ± 0.3 nm), compared to the longer (GT)15 oligomer (most probable 5'-to-5' distance of 14.3 ± 1.1 nm). During molecular recognition, XSI reveals that dopamine elicits simultaneous axial elongation and radial constriction of adsorbed ssDNA on the SWCNT surface. Our approach using XSI to probe solution-phase morphologies of polymer-functionalized SWCNTs can be applied to yield insights into sensing mechanisms and inform future design strategies for nanoparticle-based sensors.

Diameter-Dependent Competitive Adsorption of Sodium Dodecyl Sulfate and Single-Stranded DNA on Carbon Nanotubes.

The equilibrium compositions of coatings on single-wall carbon nanotubes were spectroscopically deduced for samples dispersed in dilute sodium dodecyl sulfate (SDS) and then exposed to low concentrations of ssDNA oligomers. With all studied oligomers, displacement of the SDS tended to occur at lower ssDNA concentrations for smaller diameter nanotubes than for larger diameter ones. However, the behavior varied significantly with oligomer. For example, the diameter dependence was steeper for (TAT)4 than for (ATT)4, suggesting that interstrand head-to-tail hydrogen bonding interactions play a role in SWCNT wrapping. Concentrations of ssDNA in the range of several μg/mL displace SDS from nanotubes dispersed in 1500 μg/mL SDS solutions. This effect allows the use of coating exchange to prepare ssDNA dispersions with minimal oligomer costs. Another demonstrated use exploits the structure-dependent relative coating affinities in a simple filtration method for the diameter enrichment of SWCNT mixtures.

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.

Putative Cooperative ATP-DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly.

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

Exploring the Thermodynamics of 7-Amino Actinomycin D-Induced Single-Stranded DNA Hairpin by Spectroscopic Techniques and Computational Simulations.

NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTTnGTGG) and d(CCGAAnGAGG) (denoted as GTTn and GAAn, respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT5-7AACTD/GAA5-7AACTD hairpin structure was more stable than the corresponding GTTn-7AACTD and GAAn-7AACTD (n = 3, 7). No significant ΔG difference was observed between GTTn-7AACTD and GAAn-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTTn and GAAn with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTTn and GAAn with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTTn/GAAn and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.

Gold Nanorod Assemblies: The Roles of Hot-Spot Positioning and Anisotropy in Plasmon Coupling and SERS.

Plasmon-coupled colloidal nanoassemblies with carefully sculpted “hot-spots” and intense surface-enhanced Raman scattering (SERS) are in high demand as photostable and sensitive plasmonic nano-, bio-, and chemosensors. When maximizing SERS signals, it is particularly challenging to control the hot-spot density, precisely position the hot-spots to intensify the plasmon coupling, and introduce the SERS molecule in those intense hot-spots. Here, we investigated the importance of these factors in nanoassemblies made of a gold nanorod (AuNR) core and spherical nanoparticle (AuNP) satellites with ssDNA oligomer linkers. Hot-spot positioning at the NR tips was made possible by selectively burying the ssDNA in the lateral facets via controlled Ag overgrowth while retaining their hybridization and assembly potential at the tips. This strategy, with slight alterations, allowed us to form nanoassemblies that only contained satellites at the NR tips, i.e., directional anisotropic nanoassemblies; or satellites randomly positioned around the NR, i.e., nondirectional nanoassemblies. Directional nanoassemblies featured strong plasmon coupling as compared to nondirectional ones, as a result of strategically placing the hot-spots at the most intense electric field position of the AuNR, i.e., retaining the inherent plasmon anisotropy. Furthermore, as the dsDNA was located in these anisotropic hot-spots, this allowed for the tag-free detection down to ~10 dsDNA and a dramatic SERS enhancement of ~1.6 × 108 for the SERS tag SYBR gold, which specifically intercalates into the dsDNA. This dramatic SERS performance was made possible by manipulating the anisotropy of the nanoassemblies, which allowed us to emphasize the critical role of hot-spot positioning and SERS molecule positioning in nanoassemblies.

DNA Electrophoretic Mobilities in High Ionic Strength Solutions

The free solution mobilities of small dsDNA and ssDNA oligomers have been measured as a function of ionic strength by capillary electrophoresis. The mobilities of both types of DNA decrease with increasing ionic strength, approaching limiting plateau values at high ionic strengths. The observed mobilities of the dsDNAs are similar to the mobilities calculated from the Manning electrophoresis equation, although the shapes of the mobility curves are different. The observed ssDNA mobilities cannot be described by the Manning electrophoresis equation using a single value of the axial charge spacing.

Oligomer Length Defines the Self-Assembly of Single-Stranded DNA-Collagen Complex Fibers.

Collagen and single-stranded DNA (ssDNA) complex to self-assemble into fibers depending on the length of the ssDNA and the relative amounts of collagen and ssDNA in solution. We report for the first time that when monodisperse, random sequences of ssDNA in the range of 15-90 nucleotides and type I collagen were mixed together at room temperature, fibers several tens of micrometers in length and as large as 10 μm in diameter were formed. Fiber formation was rapid and spontaneous, requiring no further treatment after mixing. Most notably, more ssDNA oligomers were incorporated into the fibers formed using shorter ssDNA oligomers. Endothelial cells formed angiogenic-like structures using the fibers with elevated expression of von Willebrand factor for cells in direct contact with the fibers. These fibers open the door to future applications in the administration and functionality of ssDNA and collagen.

(Invited) Distinct Coating Displacement Kinetics for Single-Wall Nanotube Enantiomers

It is known that specific oligomers of single-stranded DNA (ssDNA) can show selectivity toward different (n,m) structural species of single-wall carbon nanotubes (SWCNTs). These selective interactions can also extend to the left- and right-handed helical forms of a single (n,m) species. An example of this is the ability of the (ATT)4 oligo to strongly distinguish between the two optical isomers of (7,5) SWCNTs. We show that this helicity-selective coating interaction causes a resolvable shift between the two enantiomers’ fluorescence spectra. Most strikingly, the room temperature rates of displacement of the (ATT)4 coating on exposure to sodium deoxycholate are found to differ by two orders of magnitude. This is shown from measurements of time dependent fluorescence spectra. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results demonstrate the diverse and dramatic enantiomeric differences in properties of SWCNTs complexed with ssDNA.

Quantifying Mg2+ Binding to ssDNA Oligomers: A Self-Consistent Field Theory Study at Varying Ionic Strengths and Grafting Densities.

The performance of aptamer-based biosensors is crucially impacted by their interactions with physiological metal ions, which can alter their structures and chemical properties. Therefore, elucidating the nature of these interactions carries the utmost importance in the robust design of highly efficient biosensors. We investigated Mg binding to varying sequences of polymers to capture the effects of ionic strength and grafting density on ion binding and molecular reorganization of the polymer layer. The polymers are modeled as ssDNA aptamers using a self-consistent field theory, which accounts for non-covalent ion binding by integrating experimentally-derived binding constants. Our model captures the typical polyelectrolyte behavior of chain collapse with increased ionic strength for the ssDNA chains at low grafting density and exhibits the well-known re-entrant phenomena of stretched chains with increased ionic strength at high grafting density. The binding results suggest that electrostatic attraction between the monomers and Mg plays the dominant role in defining the ion cloud around the ssDNA chains and generates a nearly-uniform ion distribution along the chains containing varying monomer sequences. These findings are in qualitative agreement with recent experimental results for Mg binding to surface-bound ssDNA.

The Chemistry of Europium(III) Encountering DNA: Sprouting Unique Sequence-Dependent Performances for Multifunctional Time-Resolved Luminescent Assays.

Screening functional DNA that can fruitfully interact with metal ions is a long-standing hot topic in the fields of biotechnology, medicine, and DNA-based sensors. In this paper, we focus on the chemistry of europium(III) (Eu)coupled with single-stranded DNA (ssDNA), and we innovatively unveil that cytosine- and thymine-rich ssDNA oligomers (e.g., C16 and T16) can be effective antenna ligands to sensitize the luminescence of Eu. Luminescence lifetime spectroscopy, circular dichroic (CD) spectroscopy, and isothermal titration calorimetry (ITC) have been used to systematically characterize the interaction involved between Eu and ssDNA. In light of the resultant sequence-dependent performances, the long luminescence lifetime Eu/ssDNA-based label-free and versatile probes are further devised as a pattern distinction system for time-resolved luminescent (TRL) sensing applications. The interactions of metal ions and ssDNA can distinctively shift the antenna effect of ssDNA toward Eu as accessible pattern signals. As a result, as few as two Eu/ssDNA label-free TRL probes can discriminate 17 metal ions via principal component analysis (PCA). In addition, thiols can readily capture metal ions to switch the luminescence of Eu/ssDNA probes initially altered by metal ions. Hence, four Eu/ssDNA-metal ion ensembles are demonstrated to be a powerful label-free TRL sensor array for pattern differentiation of eight thiols and even chiral recognition of cysteine enantiomers with different concentrations. Moreover, the sensitive TRL detection of thiols in biofluids can be successfully realized by using our method, promising its potential practical usage. This is the first report of a ssDNA-sensitized Eu-based TRL platform for label-free yet multifunctional background-free sensing and would open a door for sprouting of more novel lanthanide ion/DNA-relevant strategies toward widespread applications.

Enantiomers of Single-Wall Carbon Nanotubes Show Distinct Coating Displacement Kinetics.

It is known that specific oligomers of single-stranded DNA (ssDNA) can show remarkable selectivity when coating different structural species of single-wall carbon nanotubes (SWCNTs). We report that (ATT)4 ssDNA coatings strongly distinguish between the two optical isomers of (7,5) SWCNTs. This causes resolvable shifts in their fluorescence spectra and differences of 2 orders of magnitude in the room temperature rates of coating displacement, as monitored through changes in nanotube fluorescence wavelength and intensity on exposure to sodium deoxycholate. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results reveal that enantiomeric differences in SWCNTs complexed with ssDNA are more diverse and dramatic than previously recognized.

Single-stranded DNA oligomer brush structure is dominated by intramolecular interactions mediated by the ion environment.

Single-stranded DNA (ssDNA) brushes, in which ssDNA oligomers are tethered to surfaces in dense monolayers, are being investigated for potential biosensing applications. The structure of the brush can affect the selectivity and the hybridization efficiency of the device. The structure is commonly thought to result from the balance of intramolecular interactions, intermolecular interactions within the monolayer, and molecule-surface interactions. Here, we test the hypothesis that ssDNA oligomer brush structure is dominated by intramolecular interactions. We use AFM to measure the height of an ssDNA brush and molecular dynamics to simulate the end-to-end distance, both as a function of ionic strength of the surrounding solution. The brush height and the molecule end-to-end distance match quantitatively, providing evidence that the brush structure is dominated by intramolecular interactions (mediated by ions). The physical basis of the intramolecular interactions is elucidated by the simulations.

Selective Binding of Thioflavin T in Sequence‐Exchanged Single Strand DNA Oligomers and Further Interaction with Phospholipid Membranes

AbstractSelectivity of ThT towards sequence exchanged single strand DNA (ssDNA) oligomers in absence and presence of surface charged lipid vesicles has been studied in detail. The dye shows special affinity for TCTTA sequence with its –N(CH3)3 structural counterpart and π‐interaction with adenine in 5′‐CCCTCTTAACCC‐3′ (TC4). However, fluorescence spectroscopy and molecular docking suggest that the dye is perfectly aligned with the TAA sequence in 5′‐CCCTTAATCCCC‐3′ (TT4). Interactions with cationic and anionic lipid vesicles were studied to understand the biological relevance in specificity of ThT binding to ssDNA oligomers. Fluorescence intensity of ThT increases significantly due to interaction with charged liposomes along with a considerable hypsochromic shift. Interestingly, evolution of a shoulder at 465 nm was observed solely in cationic lipid membranes due to ThT‐dimer formation. It is concluded that the ssDNA bound ThT penetrates the hydrophobic bilayer of the anionic vesicles. However, they attach superficially to the cationic vesicles facilitating dimer formation. Exchange of the nucleotide sequence in ssDNA led to change in the excited state lifetime of bound ThT in anionic liposomes.

Chemical cross‐linking of doxorubicin to synthetic oligonucleotides by formaldehyde

Anthacycline antibiotics, such as doxorubicin and daunorubicin, are among the most effective anti‐cancer drugs that treat various cancers. Previous research has shown that doxorubicin and daunorubicin were efficiently cross‐linked to double‐stranded (ds) DNA in the presence of formaldehyde in a regioselective and base specific manner. In an effort to further explore the cross‐linking of anthracycline antibiotics to DNA, doxorubicin is crosslinked to a variety of synthetic oligonucleotides including sequence specific ds and single stranded (ss) DNA oligomers. Our results demonstrate that doxorubicin is able to efficiently crosslink to the N2 amino group of guanine for the ds DNA oligomers. Intriguingly, we also discovered that doxorubicin is able to crosslink to ssDNA and ssRNA specifically to guanine residues by formaldehyde. These results suggest that ssDNA and ssRNA may serve as a new delivery system for the treatment of cancer patients by doxorubicin and daunorubicin.Support or Funding Information1R15GM109254‐01A1; R25GM061347

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein.

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4-DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3-DNA complex.

Interaction of Polymers with Single-Wall Carbon Nanotubes

Polymers are widely used for postsynthesis processing, purification, and individualization of single-wall carbon nanotubes (SWNTs) in aqueous or organic solvent environments. Here, the interaction of single-stranded DNA oligomers (ssDNA) and of a polyfluorene copolymer (F8T2) with (6,5) SWNTs was investigated from desorption kinetics and in the case of ssDNA also using adsorption isotherms. Eyring analysis of desorption rate constants reveals a linear increase of activation enthalpies with ssDNA oligomer length until ΔdesH‡ saturates at (155 ± 5) kJ·mol–1 for oligomers exceeding the ssDNA Kuhn length of about 6 nm. The Gibbs energy for desorption of ΔdesG‡ = (96 ± 1) kJ·mol–1 is length-independent because of entropy–enthalpy compensation. The saturation of desorption energies at the high polymer coverages studied here is attributed to incomplete adsorption with typically no more than a single Kuhn segment of a polymer attached to a SWNT.

Highly Sensitive and Selective Surface-Enhanced Raman Spectroscopy Label-free Detection of 3,3',4,4'-Tetrachlorobiphenyl Using DNA Aptamer-Modified Ag-Nanorod Arrays.

An improved label-free approach for highly sensitive and selective detection of 3,3',4,4'-tetrachlorobiphenyl (PCB-77), a type of polychlorinated biphenyl, via surface-enhanced Raman spectroscopy (SERS) using DNA aptamer-modified Ag-nanorod arrays as the effective substrate is reported. The devised system consists of Ag-nanorod (Ag-NR) arrays with the PCB-77 binding aptamers anchored covalently to the Ag surfaces through a thiol linker. The aptamers are made of single-stranded DNA (ssDNA) oligomers, with one end standing on the Ag surface, and upon conjugation with PCB-77, the ssDNA molecules can change their conformation to hairpin loops, so that the Raman intensity of guanines at the other end of the DNA strand increases accordingly. As such, the intensity ratio I(656 cm(-1))/I(733 cm(-1)) increases concomitantly with the increase of the concentration of PCB-77, making the quantitative evaluation of trace amounts of PCB-77 attainable. Moreover, it is found that the DNA aptamer-based Ag-NR arrays can be more responsive with a lower and optimal density of the DNA molecules modified on the substrate surface, and the best sensitivity for detection of PCB-77 can be achieved with the lower detection limit approaching 3.3 × 10(-8) M. This work therefore demonstrates that the design of aptamer-modified Ag-NRs can be used as a practically promising SERS substrate for label-free trace detection of persistent organic pollutants (POPs) in the environment.

Effects of protein-DNA complex formation on the intermolecular vibrational density of states of interfacial water.

Single-stranded DNAs (ss-DNAs) are formed as intermediates during DNA metabolic processes. ss-DNA binding (SSB) proteins specifically bind to the single-stranded segments of the DNA and protect it from being degraded. We have performed room temperature molecular dynamics simulations of the aqueous solution of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element (FUSE) binding protein (FBP) complexed with two ss-DNA oligomers. Efforts have been made to explore the influence of complex formation on low-frequency vibrational density of states of the surface water molecules. It is revealed that increased back scattering of water confined around the complexed structures leads to significant blue shifts of the band corresponding to the O···O···O bending or restricted transverse motions of water, the effect being more for the bridged water molecules. Importantly, it is demonstrated that the formation of such complexed structures of a similar type may often influence the transverse and longitudinal degrees of freedom of the surrounding water molecules in a nonuniform manner.