Single-stranded DNA Research Articles

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

While eukaryotic Argonautes play a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation (1). Here, we elucidate the activation pathway of the DNA Defense Module DdmDE system, which rapidly eliminates small, multicopy plasmids from Vibrio cholerae Seventh Pandemic Strain (7PET) (2). Through a combination of cryo-EM, biochemistry and in vivo plasmid clearance assays, we demonstrate DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that DdmD transitions from an autoinhibited, dimeric protein to monomers upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgo utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Photocatalytic colorimetry and fluorescence dual-signal biosensor for instant determination of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a photocatalytic colorimetry and fluorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/μL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1,DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

Construction of lymph nodes-targeting tumor vaccines by using the principle of DNA base complementary pairing to enhance anti-tumor cellular immune response

Tumor vaccines, a crucial immunotherapy, have gained growing interest because of their unique capability to initiate precise anti-tumor immune responses and establish enduring immune memory. Injected tumor vaccines passively diffuse to the adjacent draining lymph nodes, where the residing antigen-presenting cells capture and present tumor antigens to T cells. This process represents the initial phase of the immune response to the tumor vaccines and constitutes a pivotal determinant of their effectiveness. Nevertheless, the granularity paradox, arising from the different requirements between the passive targeting delivery of tumor vaccines to lymph nodes and the uptake by antigen-presenting cells, diminishes the efficacy of lymph node-targeting tumor vaccines. This study addressed this challenge by employing a vaccine formulation with a tunable, controlled particle size. Manganese dioxide (MnO2) nanoparticles were synthesized, loaded with ovalbumin (OVA), and modified with A50 or T20 DNA single strands to obtain MnO2/OVA/A50 and MnO2/OVA/T20, respectively. Administering the vaccines sequentially, upon reaching the lymph nodes, the two vaccines converge and simultaneously aggregate into MnO2/OVA/A50-T20 particles through base pairing. This process enhances both vaccine uptake and antigen delivery. In vitro and in vivo studies demonstrated that, the combined vaccine, comprising MnO2/OVA/A50 and MnO2/OVA/T20, exhibited robust immunization effects and remarkable anti-tumor efficacy in the melanoma animal models. The strategy of controlling tumor vaccine size and consequently improving tumor antigen presentation efficiency and vaccine efficacy via the DNA base-pairing principle, provides novel concepts for the development of efficient tumor vaccines.Graphical

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

A surface-enhanced Raman scattering and colorimetric dual-mode aptasensor for ultrasensitive detection of kanamycin based on DNA hydrogel network fishing the MIL-101@AuNP nanohybrids

It is of utmost importance to develop a simple, sensitive, and dependable technique for detecting kanamycin (Kana). Utilizing MIL-101@AuNP nanohybrids and DNA hydrogel, we devised a dual-mode aptasensor for Kana detection employing surface-enhanced Raman spectroscopy (SERS) and a colorimetric method. The MIL-101@AuNP nanohybrids displayed excellent peroxidase-like and SERS activities. In the existence of Kana, the aptamer specifically bound to Kana, releasing complementary DNA strands (cDNA). Employing free cDNA as a primer, circular DNA templates were generated for the rolling circle amplification (RCA) reaction. DNA hydrogel network formed via the physical entanglement and hybridization of two ultralong single-strand DNA chains from the double RCA-generated, capturing MIL-101@AuNP nanohybrids, thereby enhancing the SERS signal of the DNA hydrogel. Concurrently, the absorbance signal of 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system catalyzed by unfished MIL-101@AuNP was weakened. The dual-mode aptasensor, employing SERS and colorimetric methods, quantitatively detected Kana within concentration ranges of 0.003–300 ng/mL and 0.03–100 ng/mL, respectively, with detection limits (LOD) of 0.001 ng/mL and 0.018 ng/mL, respectively. Importantly, this method was applied to detect Kana-spiked milk samples, yielding satisfactory spiked recoveries. The proposed approach combines the advantages of SERS and colorimetric methods, offering high sensitivity, reliability, and broad applicability, thereby presenting a novel thinking for constructing multimode sensing platforms.

Electronic Properties of DNA Origami Nanostructures Revealed by In Silico Calculations.

DNA origami is a pioneering approach for producing complex 2- or 3-D shapes for use in molecular electronics due to its inherent self-assembly and programmability properties. The electronic properties of DNA origami structures are not yet fully understood, limiting the potential applications. Here, we conduct a theoretical study with a combination of molecular dynamics, first-principles, and charge transmission calculations. We use four separate single strand DNAs, each having 8 bases (4 × G4C4 and 4 × A4T4), to form two different DNA nanostructures, each having two helices bundled together with one crossover. We also generated double-stranded DNAs to compare electronic properties to decipher the effects of crossovers and bundle formations. We demonstrate that density of states and band gap of DNA origami depend on its sequence and structure. The crossover regions could reduce the conductance due to a lack of available states near the HOMO level. Furthermore, we reveal that, despite having the same sequence, the two helices in the DNA origami structure could exhibit different electronic properties, and electrode position can affect the resulting conductance values. Our study provides better understanding of the electronic properties of DNA origamis and enables us to tune these properties for electronic applications such as nanowires, switches, and logic gates.

Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

Co-prevalence of Human Parvovirus 4 and Primate Erythroparvovirus 1 (B19) in Sickle Cell Disease and Healthy Populations

Co-prevalence of Human Parvovirus 4 and Primate Erythroparvovirus 1 (B19) in Sickle Cell Disease and Healthy Populations

Persistent Chirality-Induced Spin-Selectivity Effect in Circular Helix Molecules.

The spin-orbit coupling (SOC), the dynamics of the nonequilibrium transport process, and the breaking of time-reversal and space-inversion symmetries have been regarded as key factors for the emergence of chirality-induced spin selectivity (CISS) and chirality-dependent spin currents in helix molecules. In this work, we demonstrated the generation of persistent CISS currents in various circular single-stranded DNAs and 310-helix proteins for the first time, regardless of whether an external magnetic flux is applied or not. This new CISS effect presents only in equilibrium transport processes, distinct from the traditional CISS observed in nonequilibrium transport processes and linear helix molecules; we term it as the PCISS effect. Notably, PCISS manifests irrespective of whether the SOC is chirality-driven or stems from heavy-metal substrates, making it an efficient way to generate chirality-locked pure spin currents. Our research establishes a novel paradigm for examining the underlying physics of the CISS effect.

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal-Free Electro-Stain Composed of DNA-Binding Proteins and Synthetic Polymers.

This paper presents the first scanning electron microscopy (SEM)-based DNA imaging in biological samples. This novel approach incorporates a metal-free electro-stain reagent, formulated by combining DNA-binding proteins and synthetic polymers to enhance the visibility of 2-nm-thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high-resolution imaging of various DNA structures including linear, circular, single-stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence-specifically incorporated into DNA molecules, which highlights the potential for SEM-based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro-stain offers electron microscopic resolution with the ease of optical microscopy.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA

BackgroundCRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. ResultsHere, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3′ end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SignificanceThe one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active Double-Stranded DNA.

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

An Overview Of The Biological Methods Used To Assess DNA Damage In Humans

The purpose of this work is to summarize the biological methods available for assessing DNA damage in humans at present. There are several methods for determining single-strand DNA breaks, alkali-labile sites, and crosslinks, including comet assays, micronucleus assays, cytogenetics (which include sister chromatid exchange and chromosomal aberration assays), DNA repair assays, oxidative DNA damage assays (measuring oxidized bases such as 8-oxo-7, 8-dihydro-2'-deoxyguanosine), and oxidized bases. There are many factors to consider when determining the best method, including how to achieve the study's objectives, what type of DNA damage to measure, and what resources are available. Combining different techniques may also contribute to a more comprehensive understanding of DNA damage and its effects on human health. By standardizing assays and advancing technology, we will be able to determine DNA damage in humans more accurately.

Extrachromosomal telomere DNA derived from excessive strand displacements

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Long-Read Nanopore-Based Sequencing of Anelloviruses

Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.

Probing the Interaction Between Biomolecules Under Sub-Zero Temperature Conditions by Electrophoresis in Ice Grain Boundaries

BackgroundPsychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established. ResultsThe chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin–biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0°C. SignificanceThis is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.