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Proceedings of the National Academy of... Oct 2023The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic...
The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the dense phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the previously most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for auto-catalytic self-replicating oligonucleotides.
Topics: Oligonucleotides; DNA; Temperature; Base Pairing
PubMed: 37847736
DOI: 10.1073/pnas.2218876120 -
Journal of Nanobiotechnology Jan 2023Dinuclear copper complexes have been designed for molecular recognition in order to selectively bind to two neighboring phosphate moieties in the backbone of double...
Dinuclear copper complexes have been designed for molecular recognition in order to selectively bind to two neighboring phosphate moieties in the backbone of double strand DNA. Associated biophysical, biochemical and cytotoxic effects on DNA were investigated in previous works, where atomic force microscopy (AFM) in ambient conditions turned out to be a particular valuable asset, since the complexes influence the macromechanical properties and configurations of the strands. To investigate and scrutinize these effects in more depth from a structural point of view, cutting-edge preparation methods and scanning force microscopy under ultra-high vacuum (UHV) conditions were employed to yield submolecular resolution images. DNA strand mechanics and interactions could be resolved on the single base pair level, including the amplified formation of melting bubbles. Even the interaction of singular complex molecules could be observed. To better assess the results, the appearance of treated DNA is also compared to the behavior of untreated DNA in UHV on different substrates. Finally, we present data from a statistical simulation reasoning about the nanomechanics of strand dissociation. This sort of quantitative experimental insights paralleled by statistical simulations impressively shade light on the rationale for strand dissociations of this novel DNA interaction process, that is an important nanomechanistic key and novel approach for the development of new chemotherapeutic agents.
Topics: Nucleic Acid Denaturation; DNA; Base Pairing; Microscopy, Atomic Force
PubMed: 36691056
DOI: 10.1186/s12951-023-01784-8 -
BioEssays : News and Reviews in... Jun 2018DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the... (Review)
Review
DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the mechanisms by which these enzymes carry out the function for which they are named. Most current views are based on inferences from crystal structures. A prominent view is that the canonical ATPase motor exerts a force on the ssDNA resulting in "pulling" the duplex across a "pin" or "wedge" in the enzyme leading to a mechanical separation of the two DNA strands. In such models, DNA base pair separation is tightly coupled to ssDNA translocation of the motors. However, recent studies of the Escherichia coli RecBCD helicase suggest an alternative model in which DNA base pair melting and ssDNA translocation occur separately. In this view, the enzyme-DNA binding free energy is used to melt multiple DNA base pairs in an ATP-independent manner, followed by ATP-dependent translocation of the canonical motors along the newly formed ssDNA tracks. Repetition of these two steps results in processive DNA unwinding. We summarize recent evidence suggesting this mechanism for RecBCD helicase action.
Topics: Adenosine Triphosphatases; Base Pairing; DNA; DNA Helicases; Escherichia coli; Escherichia coli Proteins; Translocation, Genetic
PubMed: 29603305
DOI: 10.1002/bies.201800009 -
Formation of non-base-pairing DNA microgels using directed phase transition of amphiphilic monomers.Nucleic Acids Research Apr 2022Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level...
Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level interaction of DNA is a foundational and powerful mechanism to build DNA structures at the nanoscale; however, its temperature sensitivity and weak interaction force remain a barrier for the facile and scalable assembly of DNA structures toward higher-order structures. We conducted this study to provide an alternative, non-base-pairing approach to connect nanoscale DNA units to yield micrometer-sized gels based on the sequential phase transition of amphiphilic unit structures. Strong electrostatic interactions between DNA nanostructures and polyelectrolyte spermines led to the formation of giant phase-separated aggregates of monomer units. Gelation could be initiated by the addition of NaCl, which weakened the electrostatic DNA-spermine interaction while attractive interactions between cholesterols created stable networks by crosslinking DNA monomers. In contrast to the conventional DNA gelation techniques, our system used solid aggregates as a precursor for DNA microgels. Therefore, in situ gelation could be achieved by depositing aggregates on the desired substrate and subsequently initiating a phase transition. Our approach can expand the utility and functionality of DNA hydrogels by using more complex nucleic acid assemblies as unit structures and combining the technique with top-down microfabrication methods.
Topics: Base Pairing; DNA; Hydrogels; Microgels; Nanostructures
PubMed: 35390157
DOI: 10.1093/nar/gkac232 -
Current Issues in Molecular Biology Jan 2000An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high... (Review)
Review
An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
Topics: Alleles; Base Pairing; Binding, Competitive; DNA Primers; Nucleic Acid Denaturation; Nucleic Acid Renaturation; Point Mutation; Polymerase Chain Reaction
PubMed: 11464917
DOI: No ID Found -
Accounts of Chemical Research Apr 2023RNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which...
RNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which involve treating an RNA with a reagent that modifies conformationally dynamic nucleotides, have broadly enabled examination of short- and long-range RNA structure in diverse contexts, including in living cells. For decades, chemical probing experiments have been interpreted in a per-nucleotide way, such that the reactivity measured at each nucleotide reports the average structure at a position over all RNA molecules within a sample. However, there are numerous important cases where per-nucleotide chemical probing falls short, including for RNAs that are bound by proteins, RNAs that form complex higher order structures, and RNAs that sample multiple conformations.Recent experimental and computational innovations have started a revolution in RNA structure analysis by transforming chemical probing into a massively parallel, single-molecule experiment. Enabled by a specialized reverse transcription strategy called mutational profiling (MaP), multiple chemical modification events can be measured within individual RNA molecules. Nucleotides that communicate structurally through direct base pairing or large-scale folding-unfolding transitions will react with chemical probes in a correlated manner, thereby revealing structural complexity hidden to conventional approaches. These single-molecule correlated chemical probing (smCCP) experiments can be interpreted to directly identify nucleotides that base pair (the PAIR-MaP strategy) and to reveal long-range, through-space structural communication (RING-MaP). Correlated probing can also define the thermodynamic populations of complex RNA ensembles (DANCE-MaP). Complex RNA-protein networks can be interrogated by cross-linking proteins to RNA and measuring correlations between cross-linked positions (RNP-MaP).smCCP thus visualizes RNA secondary and higher-order structure with unprecedented accuracy, defining novel structures, RNA-protein interaction networks, time-resolved dynamics, and allosteric structural switches. These strategies are not mutually exclusive; in favorable cases, multiple levels of RNA structure ─ base pairing, through-space structural communication, and equilibrium ensembles ─ can be resolved concurrently. The physical experimentation required for smCCP is profoundly simple, and experiments are readily performed in cells on RNAs of any size, including large noncoding RNAs and mRNAs. Single-molecule correlated chemical probing is paving the way for a new generation of biophysical studies on RNA in living systems.
Topics: Nucleic Acid Conformation; RNA; Base Pairing; RNA, Messenger; Nucleotides; Proteins
PubMed: 36917683
DOI: 10.1021/acs.accounts.2c00782 -
Molecular Cell Jul 2016RNA structure is intimately related to function, yet methods to identify base-paired RNA strands in a transcriptome-wide manner in cells have remained elusive. One... (Review)
Review
RNA structure is intimately related to function, yet methods to identify base-paired RNA strands in a transcriptome-wide manner in cells have remained elusive. One recent paper in Cell and two in Molecular Cell describe related methods to identify RNA sequences that interact in living cells, setting the stage for breakthroughs in our understanding of RNA structure and function.
Topics: Animals; Base Pairing; Humans; Nucleic Acid Conformation; RNA; Structure-Activity Relationship
PubMed: 27447984
DOI: 10.1016/j.molcel.2016.07.001 -
Methods (San Diego, Calif.) Mar 2009Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the... (Review)
Review
Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found in greatest abundance in the helical segments in a set of well-resolved crystal structures, including (i-ii) the canonical A.U and G.C Watson-Crick base pairs, (iii) the G.U wobble pair, (iv) the sheared G.A pair, (v) the A.U Hoogsteen pair, (vi) the U.U wobble pair, and (vii) the G.A Watson-Crick-like pair. The non-canonical pairs stand out from the canonical associations in terms of apparent deformability, spanning a broader range of conformational states as measured by the six rigid-body parameters used to describe the spatial arrangements of the interacting bases, the root-mean-square deviations of the base-pair atoms, and the fluctuations in hydrogen-bonding geometry. The deformabilties, the modes of base-pair deformation, and the preferred sites of occurrence depend on sequence. We also characterize the positioning and overlap of the base pairs with respect to the base pairs that stack immediately above and below them in double-helical fragments. We incorporate the observed positions of the bases, base pairs, and intervening phosphorus atoms in models to predict the effects of the non-canonical interactions on overall helical structure.
Topics: Base Pairing; Databases, Nucleic Acid; Hydrogen Bonding; MicroRNAs; Models, Molecular; RNA, Double-Stranded
PubMed: 19150407
DOI: 10.1016/j.ymeth.2008.12.003 -
RNA (New York, N.Y.) Oct 2019H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing...
Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.
H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs.
Topics: Base Pairing; Kinetics; Pseudouridine; Ribonucleoproteins, Small Nucleolar; Substrate Specificity; RNA, Guide, CRISPR-Cas Systems
PubMed: 31311819
DOI: 10.1261/rna.071043.119 -
Molecules (Basel, Switzerland) Sep 2021Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing... (Review)
Review
Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing their affinity and specificity while restricting their conformational space. Among them, we review our approach consisting of modifications of the 5'-C-position of the nucleoside sugar. This allows the introduction of an additional chemical handle at any position on the nucleotide chain without disturbing the Watson-Crick base-pairing. We show that 5'-C bromo or propargyl convertible nucleotides (CvN) are accessible in pure diastereoisomeric form, either for nucleophilic displacement or for CuAAC conjugation. Alternatively, the 5'-carbon can be connected in a stereo-controlled manner to the phosphate moiety of the nucleotide chain to generate conformationally constrained nucleotides (CNA). These allow the precise control of the sugar/phosphate backbone torsional angles. The consequent modulation of the nucleic acid shape induces outstanding stabilization properties of duplex or hairpin structures in accordance with the preorganization concept. Some biological applications of these distorted oligonucleotides are also described. Effectively, the convertible and the constrained approaches have been merged to create constrained and convertible nucleotides (CNA) providing unique tools to functionalize and stabilize nucleic acids.
Topics: Base Pairing; Deoxyribose; Models, Molecular; Nucleic Acid Conformation; Nucleotides
PubMed: 34641475
DOI: 10.3390/molecules26195925