Duplex Formation Research Articles

Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding.

Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level. In the past three decades, there has been a large increase in the application of single-molecule approaches to investigate helicases. These techniques, such as optical tweezers and single-molecule fluorescence, offer capabilities to monitor helicase motions with unprecedented spatiotemporal resolution, to apply quantitative forces to probe the chemo-mechanical activities of these motors and to resolve helicase heterogeneity at the single-molecule level. In this chapter, we describe a single-molecule method that combines optical tweezers with confocal fluorescence microscopy to study helicase-catalyzed DNA unwinding. Using Bloom syndrome protein (BLM), a multifunctional helicase that maintains genome stability, as an example, we show that this method allows for the simultaneous detection of displacement, force and fluorescence signals of a single DNA molecule during unwinding in real time, leading to the discovery of a distinct bidirectional unwinding mode of BLM that is activated by a single-stranded DNA binding protein called replication protein A (RPA). We provide detailed instructions on how to prepare two DNA templates to be used in the assays, purify the BLM and RPA proteins, perform single-molecule experiments, and acquire and analyse the data.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

The rise and demise of deep accretionary wedges: A long-term field and numerical modeling perspective

Abstract Several decades of field, geophysical, analogue, and numerical modeling investigations have enabled documentation of the wide range of tectonic transport processes in accretionary wedges, which constitute some of the most dynamic plate boundary environments on Earth. Active convergent margins can exhibit basal accretion (via underplating) leading to the formation of variably thick duplex structures or tectonic erosion, the latter known to lead to the consumption of the previously accreted material and eventually the forearc continental crust. We herein review natural examples of actively underplating systems (with a focus on circum-Pacific settings) as well as field examples highlighting internal wedge dynamics recorded by fossil accretionary systems. Duplex formation in deep paleo–accretionary systems is known to leave in the rock record (1) diagnostic macro- and microscopic deformation patterns as well as (2) large-scale geochronological characteristics such as the downstepping of deformation and metamorphic ages. Zircon detrital ages have also proved to be a powerful approach to deciphering tectonic transport in ancient active margins. Yet, fundamental questions remain in order to understand the interplay of forces at the origin of mass transfer and crustal recycling in deep accretionary systems. We address these questions by presenting a suite of two-dimensional thermo-mechanical experiments that enable unravelling the mass-flow pathways and the long-term distribution of stresses along and above the subduction interface as well as investigating the importance of parameters such as fluids and slab roughness. These results suggest the dynamical instability of fluid-bearing accretionary systems causes either an episodic or a periodic character of subduction erosion and accretion processes as well as their topographic expression. The instability can be partly deciphered through metamorphic and strain records, thus explaining the relative scarcity of paleo–accretionary systems worldwide despite the tremendous amounts of material buried by the subduction process over time scales of tens or hundreds of millions of years. We finally stress that the understanding of the physical processes at the origin of underplating processes as well as the forearc topographic response paves the way for refining our vision of long-term plate-interface coupling as well as the rheological behavior of the seismogenic zone in active subduction settings.

Calculation of Energy for RNA/RNA and DNA/RNA Duplex Formation by Molecular Dynamics Simulation

The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the NA-based constructs. Modern advances in computer modeling methods provide the feasibility of these calculations. We have analyzed the possibility of calculating the energy of DNA/RNA and RNA/RNA duplex formation using representative sets of complexes (65 and 75 complexes, respectively). We used the classical molecular dynamics (MD) method, the MMPBSA or MMGBSA approaches to calculate the enthalpy (ΔH°) component, and the quasi-harmonic approximation (Q-Harm) or the normal mode analysis (NMA) methods to calculate the entropy (ΔS°) contribution to the Gibbs energy (Delta G_{{37}}^{^circ } ) of the NA complex formation. We have found that the MMGBSA method in the analysis of the MD trajectory of only the NA duplex and the empirical linear approximation allow calculation of the enthalpy of formation of the DNA, RNA, and hybrid duplexes of various lengths and GC content with an accuracy of 8.6%. Within each type of complex, the combination of rather efficient MMGBSA and Q-Harm approaches being applied to the trajectory of only the bimolecular complex makes it possible to calculate the Delta G_{{37}}^{^circ } of the duplex formation with an error value of 10%. The high accuracy of predictive calculation for different types of natural complexes (DNA/RNA, DNA/RNA, and RNA/RNA) indicates the possibility of extending the considered approach to analogs and derivatives of nucleic acids, which gives a fundamental opportunity in the future to perform rational design of new types of NA-targeted sequence-specific compounds.

Contact pairs of RNA with magnesium ions-electrostatics beyond the Poisson-Boltzmann equation

The electrostatic interaction of RNA with its aqueous environment is most relevant for defining macromolecular structure and biological function. The attractive interaction of phosphate groups in the RNA backbone with ions in the water environment leads to the accumulation of positively charged ions in the first few hydration layers around RNA. Electrostatics of this ion atmosphere and the resulting ion concentration profiles have been described by solutions of the nonlinear Poisson-Boltzmann equation and atomistic molecular dynamics (MD) simulations. Much less is known on contact pairs of RNA phosphate groups with ions at the RNA surface, regarding their abundance, molecular geometry, and role in defining RNA structure. Here, we present a combined theoretical and experimental study of interactions of a short RNA duplex with magnesium (Mg2+) ions. MD simulations covering a microsecond time range give detailed hydration geometries as well as electrostatics and spatial arrangements of phosphate-Mg2+ pairs, including both pairs in direct contact and separated by a single water layer. The theoretical predictions are benchmarked by linear infrared absorption and nonlinear two-dimensional infrared spectra of the asymmetric phosphate stretch vibration which probes both local interaction geometries and electric fields. Contact pairs of phosphate groups and Mg2+ ions are identified via their impact on the vibrational frequency position and line shape. A quantitative analysis of infrared spectra for a range of Mg2+-excess concentrations and comparison with fluorescence titration measurements shows that on average 20–30% of the Mg2+ ions interacting with the RNA duplex form contact pairs. The experimental and MD results are in good agreement. In contrast, calculations based on the nonlinear Poisson-Boltzmann equation fail in describing the ion arrangement, molecular electrostatic potential, and local electric field strengths correctly. Our results underline the importance of local electric field mapping and molecular-level simulations to correctly account for the electrostatics at the RNA-water interface.

Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry.

An accurate molecular mechanics force field forms the basis of Molecular Dynamics simulations to obtain a realistic view of the structure and dynamics of biomolecules such as DNA. Although frequently updated to improve agreement with available experimental data, DNA force fields still rely in part on parameters introduced more than 20 years ago. We have developed an entirely new DNA force field, Tumuc1, derived from quantum mechanical calculations to obtain a consistent set of bonded parameters and partial atomic charges. The performance of the force field was extensively tested on a variety of DNA molecules. It excels in accuracy of B-DNA simulations but also performs very well on other types of DNA structures and structure formation processes such as hairpin folding, duplex formation, and dynamics of DNA-protein complexes. It can complement existing force fields in order to provide an increasingly accurate description of the structure and dynamics of DNA during simulation studies.

Transient Dissipative Optical Properties of Aggregated Au Nanoparticles, CdSe/ZnS Quantum Dots, and Supramolecular Nucleic Acid-Stabilized Ag Nanoclusters.

Transient, dissipative, aggregation and deaggregation of Au nanoparticles (NPs) or semiconductor quantum dots (QDs) leading to control over their transient optical properties are introduced. The systems consist of nucleic acid-modified pairs of Au NPs or pairs of CdSe/ZnS QDs, an auxiliary duplex L1/T1, and the nicking enzyme Nt.BbvCI as functional modules yielding transient aggregation/deaggregation of the NPs and dynamically controlling over their optical properties. In the presence of a fuel strand L1', the duplex L1/T1 is separated, leading to the release of T1 and the formation of duplex L1/L1'. The released T1 leads to aggregation of the Au NPs or to the T1-induced G-quadruplex bridged aggregated CdSe/ZnS QDs. Biocatalytic nicking of the L1/L1' duplex fragments L1' and the released L1 displaces T1 bridging the aggregated NPs or QDs, resulting in the dynamic recovery of the original NPs or QDs modules. The dynamic aggregation/deaggregation of the Au NPs is followed by the transient interparticle plasmon coupling spectral changes. The dynamic aggregation/deaggregation of the CdSe/ZnS QDs is probed by following the transient chemiluminescence generated by the hemin/G-quadruplexes bridging the QDs and by the accompanying transient chemiluminescence resonance energy transfer proceeding in the dynamically formed QDs aggregates. A third system demonstrating transient, dissipative, luminescence properties of a reaction module consisting of nucleic acid-stabilized Ag nanoclusters (NCs) is introduced. Transient dynamic formation and depletion of the supramolecular luminescent Ag NCs system via strand displacement accompanied by a nicking process are demonstrated.

Amplification-free detection of bacterial genes using a signaling probe-based DNA microarray

In this study, we developed a novel DNA microarray system that does not require fluorophore-labeling, amplification, or washing of the target nucleic acid fragments. Two types of DNA probes (so-called “signaling probes”) labeled with a fluorescence dye (Cy3) and quencher molecule (BHQ2) were spotted on the DNA microarray such that fluorescent signals of Cy3 could be quenched by BHQ2 due to duplex formation between the probes. The addition of the target DNA or RNA fragments disrupted the duplex formed by the probes, resulting in the generation of fluorescence signals. We examined the assay conditions of the signaling probe-based DNA microarray, including the design of the probes, hybridization temperatures, and methods for fragmentation of target molecules. Since this approach does not require time-consuming processes, including labeling, amplification, and washing, the assay achieved specific detection of 16S rDNA and 16S rRNA extracted from Escherichia coli within 60 min, which was significantly rapid compared to conventional PCR-dependent DNA microarrays.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS – CoV-2

ABSTRACT Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3ʹ proximal genome body sequence to a 5ʹ untranslated leader sequence. This leads to a fusion between the common 5ʹ leader sequence and a 3ʹ proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity.

We demonstrate the ability to distinguish Pb2+ from K+ within the central cavity of the antiparallel G-quadruplex (GQ) DNA produced by the thrombin binding aptamer (TBA) using an internal molecular rotor fluorescent probe. An indole-aldehyde containing an acyclic N-glycol group was first employed in the on-strand Knoevenagel condensation with five different heterocyclic quaternary cationic acceptors to assess the molecular rotor character of the resulting cyanine-styryl dyes within duplex DNA. An indole-pyridinium (4PI) nucleobase surrogate displayed the greatest turn-on emission response to duplex formation and was thus inserted into the loop residues of TBA to monitor GQ-folding in the presence of Pb2+ versus K+. TBA-4PI exhibits turn-on emission upon Pb2+-binding with a brightness (ε·Φfl) of 9000 cm-1 M-1 compared to K+-binding (ε·Φfl ∼ 2000 cm-1 M-1) due to Pb2+-induced GQ rigidity with 4PI-G-tetrad stacking interactions. The Pb2+-bound TBA-4PI GQ also provides energy-transfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb2+ from K+ within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb2+ ions with an apparent dissociation constant (Kd) of 63 nM and a limit of detection (LOD) of 19 nM in an aqueous buffer. It can also distinguish Pb2+ (230 nM) from K+ (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.

HOXC11 positively regulates the long non-coding RNA HOTAIR and is associated with poor prognosis in colon adenocarcinoma.

Colorectal cancer ranks third in terms of incidence and second in terms of mortality worldwide. The homeobox transcript antisense intergenic RNA (HOTAIR), which was found to be located on the antisense chain of the homeobox C (HOXC) gene cluster, is a long non-coding RNA involved in multiple types of tumors. The role of HOXC11 in tumors remains unclear. Reverse transcription-quantitative PCR was performed to detect the expression level of HOXC11 in colon adenocarcinoma. Cell proliferation and invasion were assessed. RNase protection assay was used to test the possibility of RNA duplex formation. The increased expression and co-expression trend of HOXC11 and HOTAIR were identified in multiple types of cancer from The Cancer Genome Atlas and the results were validated in 12 colon adenocarcinoma and paired non-tumor tissue samples. The expression of HOXC11 and HOTAIR was found to be associated with poor prognosis in colon adenocarcinoma and kidney renal clear cell carcinoma. Furthermore, HOXC11 was found to positively regulate HOTAIR by RNA duplex formation and promoted the proliferation and invasion of colon adenocarcinoma cells.

Photo-Controllable Phase Transition of Arylazopyrazole-Conjugated Oligonucleotides.

Phase transition is a promising aspect of DNA as biopolymers. Anionic DNA oligonucleotides easily form complexes with cationic polypeptides such as polylysine, and duplex formation significantly influences their complexation and resulting microcompartments. In this study, phase transition of microcompartments containing DNA and polylysine was systematically induced by modulating duplex formation of arylazopyrazole-conjugated oligonucleotides with light. We demonstrated that UV irradiation destabilized DNA duplex and generated isotropic coacervates, while duplex stabilization by visible light irradiation caused the formation of liquid crystalline coacervates. This photocontrol of phase transition was highly repeatable, and similar changes were observed even after ten cycles of light irradiation. Our approach would provide a robust control layer to the development of tailor-made microcompartments.

Investigation on the competition of duplex/ G-quadruplex/ i-motif in telomere sequences and c-MYC gene with a solid-state nanopore sensor

Apart from duplex helix, the coexisted noncanonical DNA may be involved in inhibiting DNA replication, and controlling gene expression. The competition of duplex/ G4/ i-motif depends on many factors, including sequence information, external conditions and binding effects. This work looked into the human telomere sequences of different length (hTelo-1, 2) and intramolecular G-rich and C-rich strands (hTelo-3, 4, 5). Nanopore translocation properties suggested that in the presence of their complementary sequences in the mixed K+/Cs+ buffer, G4 was the major species, and G4 from hTelo-2 that holds multiple G-quartets formed slower than shorter hTelo-1. The intramolecular G/C-rich strands hTelo-3, 4, 5 predominantly formed duplex rather than quadruplex under K+ or acidic conditions, indicating that intramolecular quadruplex is less stable than duplex due to the strand strain. Translocation behaviors of mixed proto-oncogene cMYC/c-cMYC illustrated that G4 was formed in both K+ and Cs+, and Cs+ inhibited the duplex formation in K+/Cs+ buffer. The increase of the ratio of Cs+ will decrease the duplexes species. Our work demonstrated the relative stability of duplex/quadruplex under various conditions with distinct strands, which is useful for the investigation of DNA secondary structural dynamics and transition mechanism in biological function regions, and paved the way for the potential of disease-targeted diagnosis.

Saliva "Treat-and-Heat" Triplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for SARS-CoV-2.

The demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostics that are faster, cheaper, and simpler to run than nasopharyngeal-based reverse transcription quantitative PCR (RT-qPCR) tests remains unmet in many parts of the world. In the Philippines, geographical and economic access to quality diagnostic testing remains out of reach for many communities. We describe the preclinical development of a fluorescence-based reverse transcription loop-mediated isothermal amplification test that uses drooled saliva as the biospecimen. Six treat-and-heat ("direct") procedures that inactivate the virus and release the target RNA were compared. Using duplexed As1e and E1 primers, protocols derived from Ben-Assa et al. (2020) using proteinase K or from Rabe and Cepko (2020) using TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)/EDTA provided reliable RNA amplification. The TCEP/EDTA-based method in particular showed improvement in robustness in duplex vs. singleplex format. Inclusion of human β-actin primers provided a triplex test with an internal amplification control that could be distinguished from SARS-CoV-2 amplicons based on melt curve analysis. After including the dUTP/uracil-DNA glycosylase system and implementing laboratory procedures to avoid cross-contamination, false positive amplification was acceptably rare. The duplex or triplex tests are predicted to reliably detect patient salivary viral loads >100 copies/μL and to yield equivocal results between 10 and 100 copies/μL. These viral loads, corresponding to RT-qPCR C t ∼29-32, are expected to identify the majority of infected and, particularly, of infectious patients. If clinically validated, the test would provide additional testing capacity requiring only a fraction of the time, cost, and infrastructure of the current nasopharyngeal swab-based RT-qPCR test, thereby improving access to testing for more Filipinos.

In-Silico Analysis of rSNPs in miRNA:mRNA Duplex Involved in Insulin Signaling Genes Shows a Possible Pathogenesis of Insulin Resistance.

Insulin resistance is a condition in which the body produces insulin but is unable to use it effectively. Aberrations in insulin signaling are known to play a crucial role in the pathogenesis of this disease state. Eventually, patients will have glucose build-up in their blood instead of being absorbed by the cells, leading to type 2 diabetes. In the current study, we focus on understanding the role of rSNP mediated miRNA:mRNA dysregulation and its impact on the above metabolic condition. More than 30 genes involved in the insulin signaling pathway were found using the KEGG database. The 3'UTR end of genes was studied by using RegRNA and Ensembl, whereas TargetScan along with miRbase were used to identify their target miRNAs. Binding free energy was used as a parameter to analyze the effect of polymorphism on the miRNA:mRNA duplex formation. Further, the UNA fold was used to determine the heat capacity changes. The genes INSR, INS, GLUT4, FOXO1, IL6, TRIB3, and SREBF1, were selected for analysis. Multiple miRNAs, hsa-miR-16-5p, hsa-miR-15a-15p were identified in the SNP occurring region for INSR. INS, too, showed similar results. INSR, INS, and TRIB3 were found to have the maximum change in their binding free energy due to rSNP variation. A destabilisation in the heat capacity values was observed too, which contributed due to rSNP induction. A direct relationship between miRNA target polymorphism and the stability of the miRNA:mRNA duplex was observed. The current methodology used to study insulin resistance pathogenesis could elaborate on our existing knowledge of miRNA-mediated disease states.

On-line duplex molecularly imprinted solid-phase extraction for analysis of low-abundant biomarkers in human serum by liquid chromatography-tandem mass spectrometry

In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets – NLLGLIEAK (ProGRP) and ELPLYR (NSE) – in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R2≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.

Carbon ink-based electrodes modified with nanocomposite as a platform for electrochemical detection of HIV RNA

This work reports a new and easy-to-use methodology for construction of screen-printed electrodes (SPE) based on carbon ink and its modification with 4-aminothiophenol (4-ATP) and gold nanoparticles for detection of HIV RNA. Morphological analysis for SPE/4-ATP/AuNps+DNA probe showed increased roughness indicating that 4-ATP monolayer works as a network for connection of functionalized gold nanoparticles with specific oligonucleotide probe to the RNA target. The biosensor response was monitored by indirect electrodetection using ethidium bromide as indicator of duplex formation. The peak current response of ethidium bromide was linear related to the logarithm of concentration of HIV RNA, detecting up to 6 copies/mL and linear range of 6 to 11,000 copies/mL of RNA, covering the clinically relevant concentration range (50–200 copies/mL). The biosensor discriminates negative and positive samples, making it possible to quantify the viral load, a crucial factor for the development of biosensors for the early detection of HIV.

Use of anion-exchange HPLC to study DNA conformational polymorphism

Anion-exchange chromatography carried out under non-denaturing conditions is a versatile tool to differentiate DNA conformations. In this work, the utility of this form of HPLC was demonstrated in four examples. The hairpin and duplex forms of d(CG)9 were readily resolved, which allowed for the studies of the influence of salt on the equilibrium of these two forms of secondary structures. Similarly, the minimum size of Tn in the loop region required for the sequence 5′-d(CCCAA-(T)n-TTGGG)-3′ to form hairpin was established to be two nucleotides using anion-exchange HPLC and fluorescence resonance energy transfer. Furthermore, the efficiency of hybridization of partially self-complementary sequences d[(CG)6Nx] was readily monitored by non-denaturing anion-exchange HPLC. Finally, different structures adopted by quadruplex-forming sequences were resolved in the same manner.

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

Antisense overlapping long non-coding RNA regulates coding arachidonate 12-lipoxygenase gene by translational interference

The arachidonate 12-lipoxygenase (ALOX12) enzyme catalyzes polyunsaturated fatty acids and facilitates generation of bioactive lipid mediators associated with various biological processes and disease pathologies. The human genome assembly revealed that the ALOX12 gene overlaps an antisense non-coding gene designated as ALOX12-antisense 1 (ALOX12-AS1). This arrangement indicates that the uncharacterized ALOX12-AS1 long non-coding RNA (lncRNA) may bind to the sense coding ALOX12 mRNA to form an antisense-sense duplex providing the basis of a novel ALOX12 regulatory mechanism. Therefore, this study was designed to determine whether the interaction of ALOX12-AS1 with ALOX12 mRNA functions as an anti-sense/sense duplex-mediated regulatory mechanism controlling the cellular content of ALOX12. Our findings indicate that two major isoforms of ALOX12-AS1 lncRNA are ubiquitously expressed in a variety of primary adult human tissues and different transformed cell types. RNA-FISH revealed cell-type-specific cytosolic as well as nuclear and nucleolar localization of the lncRNA. Interestingly, phorbol ester-induced nucleo-cytoplasmic translocation of the lncRNA in monocytic THP-1 cells resulted in a reduction of ALOX12 protein without a concomitant change in its mRNA level. This indicated ALOX12-AS1 operates via an antisense-sense duplex-mediated translational downregulation mechanism. This deduction was validated by demonstrating sense/antisense duplex formation and an association of the duplex with ribosomal proteins in HEK293 cells. Overall, this study revealed a hitherto unknown mechanism of antisense lncRNA-mediated translational downregulation of ALOX12 that adds to the existing regulatory mechanisms for the modulation of potent bioactive lipid mediators that contribute to both health and disease.