Double Helix Research Articles

Calcium Alginate Encapsulation of Rice Starch, Instead of a Physical Mixture of Both, Regulates the Fermentation Rate and Production of Acetate

ABSTRACTCalcium alginate–encapsulated rice starch (AES) could be potentially applied as a rice analog with a significantly improved amount of resistant starch, while its effects on gut microbiota remain less clear. To this end, structural characteristics of AES and their impact on gut microbiota, fermentation rate, and short‐chain fatty acid (SCFA) production were examined using an in vitro batch fermentation method. Cooked AES showed a significantly higher amount of intermolecular interactions (∼46 times), short‐range double helices, and degree of crystallinity compared to the simple mixture of rice starch and calcium alginate (Mix), resulting in a more homogenous and densely packed network microstructure. As a result, AES, instead of Mix, showed a significantly slower gas production rate (∼17%), while relatively higher production of SCFAs, especially the ratio of acetate. Bifidobacterium pesudocatenulatum was possibly responsible for the higher production of acetate in AES. Collectively, these results show that AES has the potential to be used as a slowly fermentable carbohydrate, favoring the production of acetate in the human colon.

Efficient and Rapid Enrichment of Extracellular Vesicles Using DNA Nanotechnology-Enabled Synthetic Nano-Glue.

Swift and efficient enrichment and isolation of extracellular vesicles (EVs) are crucial for enhancing precise disease diagnostics and therapeutic strategies, as well as elucidating the complex biological roles of EVs. Conventional methods of isolating EVs are often marred by lengthy and laborious processes. In this study, we introduce an innovative approach to enrich and isolate EVs by leveraging the capabilities of DNA nanotechnology. We have developed a novel multivalent cholesterol-modified paranemic crossover DNA (PX-DNA-chol) construct, which is a four-stranded DNA structure containing adjacent double helices intertwined with their local helix axes parallel and serves as an effective synthetic nano-glue. This construct promotes the rapid coalescence of nanoscale EVs into clusters of micrometer scale, thereby streamlining their enrichment. Utilizing a conventional low-speed centrifuge, this intriguing methodology achieves a rapid concentration of EVs within minutes, bypassing the laborious and high-speed centrifugation steps typically required. The quality of EVs isolated by our technique is comparable to that obtained through ultracentrifugation methods. Given these advancements, our PX-DNA-chol-facilitated EVs enrichment protocol is poised to advance the field of EVs research, providing a robust and accessible tool for in-depth studies of EVs.

Double-Helix Electrode Ion Funnel: A New Ion Funnel Design with an Extended Mass Range.

The development of an atmospheric pressure interface (API) with a high ion transfer efficiency and wide mass range is advantageous for the performance improvement of mass spectrometry (MS) instruments. In this work, a novel ion guide, namely, the double-helix electrode ion funnel (DHE-IF), has been developed to enhance the ion transmission over a wide mass range in the rough vacuum region. The DHE-IF consists of two funnel-shaped helix electrodes. There are almost no potential "traps" along the central axis of DHE-IF due to the continuous double-helix electrode structure compared to the stacked ring ion funnel. The electrode design of the DHE-IF assembly was guided by ion trajectory simulations. After being fabricated, DHE-IF was integrated into an ESI-TOF-MS platform for tests. A conventional stacked ring ion funnel (IF) was also tested for comparison. The experimental results showed that DHE-IF extended the transmission window of the IF and improved the efficiency of the simultaneous transferring of low and medium m/z ions. In addition, the intensities of caffeine ions (m/z = 195) and reserpine ions (m/z = 609) were enhanced by more than 50% and 10%, respectively. These values were compared with the results obtained by the IF. The DHE-IF is expected to be widely used as an ion import device in MS instruments, which is due to its improved performance and advantages in, e.g., integration and power supply design.

Carbonless DNA.

Carbonless DNA was designed by replacing all carbon atoms in the standard DNA building blocks with boron and nitrogen, ensuring isoelectronicity. Electronic structure quantum chemistry methods (DFT(ωB97XD)/aug-cc-pVDZ) were employed to study both the individual building blocks and the larger carbon-free DNA fragments. The reliability of the results was validated by comparing selected structures and binding energies using more accurate methods such as MP2, CCSD, and SAPT2+3(CCD)δMP2. Carbonless analogs of DNA components, including cytosine, thymine, guanine, adenine, and deoxyribose, were investigated, showing strong resemblance to the carbon-based versions in terms of spatial structure, polarity, and molecular interaction capabilities. Complementary base pairs of the carbonless analogs exhibited a similar number and length of hydrogen bonds as those found in their carbon-containing counterparts, with binding energies for A-T and G-C analogs remaining comparable. Carbonless DNA fragments containing two and six base pairs were studied, revealing double-helix structures analogous to natural DNA. Structural parameters such as fragment size, hydrogen bond lengths, and rise per base pair were consistent with those observed in unmodified DNA. Docking simulations with a 12 base pair fragment and netropsin as a ligand indicated a slight shift in binding preference for the carbonless DNA through the minor groove, with an approximate 25% increase in binding affinity compared to natural DNA.

Divergent evolution of opposite base specificity and single-stranded DNA activity in animal and plant AP endonucleases.

Apurinic/apyrimidinic (AP) endonucleases are key enzymes responsible for the repair of base-less nucleotides generated by spontaneous hydrolysis or as DNA repair intermediates. APE1, the major human AP endonuclease, is a druggable target in cancer and its biological function has been extensively studied. However, the molecular features responsible for its substrate specificity are poorly understood. We show here that, in contrast to APE1, its Arabidopsis ortholog ARP (apurinic endonuclease-redox protein) exhibits orphan base-dependent activity on double-stranded DNA and very poor AP cleavage capacity on single-stranded DNA (ssDNA). We found that these differences are largely a consequence of the variation at two DNA intercalating amino acids that have undergone divergent changes in the metazoan and plant lineages. Swapping the identity of the residue invading the minor groove is sufficient to switch the orphan base specificities of APE1 and ARP. The affinity for ssDNA is largely determined by the major groove invading residue, and swapping its identity switches the ability of APE1 and ARP to cleave AP sites in ssDNA. Importantly, we show that the critical residue for ssDNA cleavage is crucial for mammalian APE1 function in antibody class switch recombination, suggesting an evolutionary advantage for ssDNA activity. These findings provide new molecular insights into the evolution of AP endonucleases.

Chiral Nanostructures from Artificial Helical Polymers: Recent Advances in Synthesis, Regulation, and Functions.

Helical structures such as right-handed double helix for DNA and left-handed α-helix for proteins in biological systems are inherently chiral. Importantly, chirality at the nanoscopic level plays a vital role in their macroscopic chiral functionalities. In order to mimic the structures and functions of natural chiral nanoarchitectures, a variety of chiral nanostructures obtained from artificial helical polymers are prepared, which can be directly observed by atomic force microscopy (AFM), scanning tunneling microscopy (STM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). This review mainly focuses on the formation of chiral nanostructures and the morphology regulation triggered by polymer chain length, concentration, solvent, temperature, photoirradiation, and chemical additives. In addition, the distinct chiral functions including chiral recognition, circularly polarized luminescence, drug release, cell imaging, and antibiosis are also discussed.

Identification and characterization of shifted G•U wobble pairs resulting from alternative protonation of RNA.

RNA can serve as an enzyme, small molecule sensor, and vaccine, and it may have been a conduit for the origin of life. Despite these profound functions, RNA is thought to have quite limited molecular diversity. A pressing question, therefore, is whether RNA can adopt novel molecular states that enhance its function. Covalent modifications of RNA have been demonstrated to augment biological function, but much less is known about non-covalent alterations such as novel protonated or tautomeric forms. Conventionally, a G•U wobble has the U shifted into the major groove. We used a cheminformatic approach to identify four structural families of shifted G•U wobbles in which the G instead resides in the major groove of RNA, which requires alternative tautomeric states of either base, or an anionic state of the U. We provide experimental support for these shifted G•U wobbles via the potent, and unconventional, in vivo reactivity of the U with dimethylsulfate (DMS) in three organisms. These shifted wobbles may play important functional roles and could serve as drug targets. Our cheminformatics approach is general and can be applied to identify alternative protonation states in other RNA motifs, as well as in DNA and proteins.

Low background electrochemical sensor based on HCR towards acute myocardial infarction-specific miRNA detection.

Acute myocardial infarction (AMI) accounts for a significant proportion of global fatalities, and early detection is crucial for improving patient outcomes. However, current diagnostic methods often struggle to detect AMI in its early stages. Herein, we present an electrochemical sensor utilizing a fractal gold (FracAu) electrode and hybridization chain reaction (HCR) amplification technology to detect AMI-specific microRNAs (miRNAs). When the target sequence was added, the HCR was triggered, leading to the formation of a long-nicked DNA double helix that efficiently captured a larger quantity of positively charged RuHex molecules, resulting in significant electrochemical signal amplification. More importantly, to avoid false positive signals, exonuclease I (Exo I) was introduced to selectively cleave single-stranded DNA (ssDNA) probes. These ssDNA probes, underwent random hydrolysis from hpDNA probes, could hybridize with helper DNA1 in the absence of the target, initiating the HCR process and producing a false positive signal. The inclusion of Exo I effectively avoided false positive signals and reduced background noise. Under optimized conditions, the fabricated sensor exhibited significant sensitivity and selectivity, showing a broad linear detection range from 10 pM to 10 nM and a low limit of 0.9 fM. The fabricated electrochemical sensor also successfully detected AMI-specific miRNA in real serum samples, underscoring its diagnostic promise. By providing a reliable tool for early detection, the innovative sensor holds significant potential in combating global cardiovascular disease-related mortality rates.

Effect of (-)-epigallocatechin gallate palmitate complexation under mild temperature on the structure and nutritional functions of porous rice starch.

The correlation among the hierarchical structure, physicochemical properties, and nutritional functions of porous rice starch after absorbing and complexing with (-)-epigallocatechin gallate palmitate (P-EGCG) under mild temperatures at different reaction times were investigated. The P-EGCG loading rate (19.6%-28.5%) of porous starch increased after hydrolysis with a mixture of amyloglucosidase and α-amylase for 3 and 6h, respectively. A decrease in the melting enthalpy of the amylopectin double helix and an increase in the melting enthalpy of the V-type helices after complexation was observed with longer reaction times. The retention index of P-EGCG after 6h of incubation was 57.11% following 21 d of storage. These structural changes significantly transformed portions of the rapidly and slowly digestible starches into resistant starch (41.68%-47.84%), accompanied by enhanced thermal stability, antioxidant activity, and enteropathogenic bacteria-inhibiting ability. Therefore, porous rice starch complexed with P-EGCG may provide controlled digestion, antioxidant activity, and potential gut microbiota benefits.

Double helix rotating TENGs driven by ultra-low loading for harvesting high-entropy water flow energy

Water flow energy in rivers and lakes is a huge clean energy source and widely distributed in nature. Its low water velocity makes it difficult for electromagnetic generators to effectively harvest this energy. Therefore, harvesting high-entropy water flow energy is of great significance to the development of distributed power supply and sensing. In this study, we present a double helix rotating triboelectric nanogenerator (DHR-TENG) that can effectively convert the ultra-low water flow energy into electrical energy. DHR-TENG is designed to optimize space utilization and has a reciprocating transmission mechanism to ensure the continuous operation, achieving a surprisingly high charge density of 356.69 μC⋅m-3 and peak power density of 11.66 W⋅m-3. Compared with similar structures reported by predecessors, the maximum elevation is about 242 times and 2 times, respectively. A stable electrical output performance was achieved by DHR-TENG at ultra-low water flow velocity of 0.4∼0.8 m/s, the harvested energy can drive self-powered temperature sensor systems and wireless signal transmission systems. This work not only effectively improves the electrical output performance at ultra-low water flow velocity, but also helps to promote environmental monitoring and provides a feasible method for establishing early warning in the environment.

Autoclaved Starch: Structure and Functionality Relationship in a Matrix With the Same Contribution ofAmylose and Amylopectin.

The rational use of autoclaved starches in food applications is difficult because there is a lack of information on their structure-functionality relationship. The novelty of this research relies on disclosing such an association. Hylon V starch was autoclaved at 105, 120, and 135°C to investigate its crystalline and double-helical features and its relationship with functionality. In autoclaved Hylon V starch, interactions of amylopectin and amylose improved while the crystalline regions decreased. The degree of double helices (DD) decreased after autoclaving at 105°C and the degree of order (DO) increased after treatment at 120 and 135°C. The water solubility index (WSI) (4.63-6.38%) and swelling power (SP) (4.39-7.1 g/g) increased when the temperature increased. On the other hand, water (103.49-225.01%) and oil (61.91-94.53%) holding capacity (WHC and OHC, respectively) increased after autoclaving treatment, although the values decreased with the treatment intensity. The functional properties were affected when the structure changed as a function of the treatment temperatures. PCA analysis showed that WSI and SP of autoclaved Hylon V starch were associated with a high DD, with better compaction, and with stronger amylopectin-amylose interactions. WHC and OHC were associated with better crystallinity, stronger interactions of amylopectin and amylose, and heterogeneous double-helical crystallites. These findings are useful for understanding the structure-functionality relationship of autoclaved Hylon V starch and pave the way for future research regarding the effects of its incorporation on the properties of food matrices such as bread, yogurt, cakes, and pudding.

Polyhydroxy starch with abundant hydroxyls and a unique structure enables uniform Zn deposition.

Zinc metal is a promising anode material for zinc-ion batteries (ZIBs), but severe side reactions and dendrite formation hinder its commercialization. In this study, starch is introduced into the ZnSO4 electrolyte for stabilizing the Zn anode. With abundant hydroxyl groups, starch can reconstruct the H-bond system in the electrolyte, suppressing side reactions. Moreover, the unique ring and double-helix structures of starch show strong interactions with Zn2+ ions. The large molecule structure also exhibits a steric hindrance effect, regulating the diffusion and reduction of Zn2+. Additionally, starch molecules can adsorb onto the Zn anode, promoting the formation of a solid-electrolyte interphase (SEI) and resulting in uniform Zn deposition along the (002) plane. Consequently, this approach enhances the stability and reversibility of the Zn anode.

Prebiotic RNA self-assembling and the origin of life: Mechanistic and molecular modeling rationale for explaining the prebiotic origin and replication of RNA.

In recent years, a great interest has been focused on the prebiotic origin of nucleic acids and life on Earth. An attractive idea is that life was initially based on an autocatalytic and autoreplicative RNA (the RNA-world). RNA duplexes are right-handed helical chains with antiparallel orientation, but the rationale for these features is not yet known. An antiparallel (inverted) stacking of purine nucleosides was reported in crystallographic studies. Molecular modeling also supports the inverted orientation of nucleosides. This preferential stacking can also appear when nucleosides are included in a montmorillonite clay matrix. Free-energy values and geometrical parameters show that D-ribose chirality is preferred for the formation of right-handed RNA molecules. Thus, a "zipper" model with antiparallel and auto-intercalated nucleosides linked by phosphate groups can be proposed to form single RNA chains. Unstacking with strand separation and base pairing by H-bonding, results in shortening and inclination of ribose-phosphate chains, leading to right-handed helicity and antiparallel duplexes. Incorporation of complementary precursors on the major groove template by a self-assembly mechanism provides a prebiotic (non-enzymatic) "tetris" replication model by formation of a transient RNA tetrad and tetraplex. Original hairpin motifs appear as simple building units that form typical RNA structures such as hammerheads, cloverleaves and dumbbells. They occur today in the circular viroids and virusoids, as well as in highly branched and complex rRNA molecules.

Torsional Vibrations of DNA and RNA

Experimental evidence of suppression of bacterial survival at resonant frequencies is given, it is indicated that centimeter waves can cause hormesis. It is shown that the torsional rigidity of DNA or RNA helices in the free and in the compactified state differs by 30 orders of magnitude. Exact formulas for the natural frequencies of torsional vibrations of DNA and RNA are obtained. The confirmations of the theory according to which the natural frequencies of torsional vibrations of DNA helices are located in the centimeter spectrum are indicated.

Unveiling the self-assembly process of gellan-chitosan complexes through a combination of atomistic simulations and experiments.

Polyelectrolyte complexes (PECs), formed via the self-assembly of oppositely charged polysaccharides, are highly valued for their biocompatibility, biodegradability, and hydrophilicity, offering significant potential for biotechnological applications. However, the complex nature and lack of insight at a molecular level into polyelectrolytes conformation and aggregation often hinders the possibility of achieving an optimal control of PEC systems, limiting their practical applications. To address this problem, an in-depth investigation of PECs microscopic structural organization is required. In this work, for the first time, a hybrid approach that combines experimental techniques with atomistic molecular dynamics simulations is used to elucidate, at a molecular level, the mechanisms underlying the aggregation and structural organization of complexes formed by gellan and chitosan, i.e. PECs commonly used in food technology. This combined analysis reveals a two-step complexation process: gellan initially self-assembles into a double-helix structure, subsequently surrounded and stabilized by chitosan via electrostatic interactions. Furthermore, these results show that complexation preserves the individual conformation and intrinsic functionality of both polyelectrolytes, thereby ensuring the efficacy of the PECs in biotechnological applications.

Recognition of Noncanonical RNA Base Pairs Using Triplex-Forming Peptide Nucleic Acids.

Noncanonical base pairs play an important role in enabling the structural and functional complexity of RNA. Molecular recognition of such motifs is challenging because of their diversity, significant deviation from the Watson-Crick structures, and dynamic behavior, resulting in alternative conformations of similar stability. Triplex-forming peptide nucleic acids (PNAs) have emerged as excellent ligands for the recognition of Watson-Crick base-paired double helical RNA. The present study extends the recognition potential of PNA to RNA helices having noncanonical GoU, AoC, and tandem GoA/AoG base pairs. The purines of the noncanonical base pairs formed M+·GoU, T·AoC, M+·GoA, and T·AoG Hoogsteen triples of similar or slightly reduced stability compared to the canonical M+·G-C and T·A-U triples. Recognition of pyrimidines was more challenging. While the P·CoA triple was only slightly less stable than P·C-G, the E nucleobase did not form a stable triple with U of the UoG wobble pair. Molecular dynamics simulations suggested the formation of expected Hoogsteen hydrogen bonds for all of the stable triples. Collectively, these results expand the scope of triple helical recognition to noncanonical structures and sequence motifs common in biologically relevant RNAs.

Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for Mtb's adaptation to the human host environment. Our study focuses on one notable Mtb repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for Mtb survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of mce3R orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the yrbE3A (mce3) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a Kd of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.

All-at-once RNA folding with 3D motif prediction framed by evolutionary information.

Structural RNAs exhibit a vast array of recurrent short 3D elements involving non-Watson-Crick interactions that help arrange canonical double helices into tertiary structures. We present CaCoFold-R3D, a probabilistic grammar that predicts these RNA 3D motifs (also termed modules) jointly with RNA secondary structure over a sequence or alignment. CaCoFold-R3D uses evolutionary information present in an RNA alignment to reliably identify canonical helices (including pseudoknots) by covariation. We further introduce the R3D grammars, which also exploit helix covariation that constrains the positioning of the mostly non-covarying RNA 3D motifs. Our method runs predictions over an almost-exhaustive list of over fifty known RNA motifs (everything). Motifs can appear in any non-helical loop region (including 3-way, 4-way and higher junctions) (everywhere). All structural motifs as well as the canonical helices are arranged into one single structure predicted by one single joint probabilistic grammar (all-at-once). Our results demonstrate that CaCoFold-R3D is a valid alternative for predicting the all-residue interactions present in a RNA 3D structure. Furthermore, CaCoFold-R3D is fast and easily customizable for novel motif discovery.

Adding a twist to the loops: the role of DNA superhelicity in the organization of chromosomes by SMC protein complexes.

Structural maintenance of chromosomes (SMC) protein complexes, including cohesin, condensin, and the Smc5/6 complex, are integral to various processes in chromosome biology. Despite their distinct roles, these complexes share two key properties: the ability to extrude DNA into large loop structures and the capacity to alter the superhelicity of the DNA double helix. In this review, we explore the influence of eukaryotic SMC complexes on DNA topology, debate its potential physiological function, and discuss new structural insights that may explain how these complexes mediate changes in DNA topology.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.