DNA-RNA Interactions Research Articles

Autochemophoretic DNA Motors Generate 100+ Piconewton Forces

DNA walkers, which “walk” along linear or planar tracks via burnt-bridge hybridization interactions, are promising synthetic analogs of motor proteins such as kinesin, myosin, and dynein. DNA walkers can precisely transport nanoscale cargo but cannot generate piconewton-scale force. This primary function of motor proteins is necessary for countless processes including muscle contraction, clotting, immunosensing, embryogenesis, and mechanosensation. We present progress towards the design of force-generating nanomachines by showing that highly polyvalent DNA motors (HPDMs) generate 100+ piconewtons of force via a novel mechanism that we term autochemophoresis. HPDMs are DNA-coated microparticles that connect to planar RNA-functionalized surfaces via DNA-RNA hybridization (Yehl & Salaita et. al., Nature Nanotechnology 2016). Ribonuclease H enables translocation by cleaving the RNA-DNA duplexes, resulting in an RNA depletion track in the HPDM's wake. Translocating for hours at micron/minute speeds, HPDMs are the fastest, most processive DNA-based motors reported to date. To test HPDMs’ force generation capability we designed a single molecule fluorescence microscopy experiment which enables direct visualization of mechanically-ruptured molecular bonds. Surprisingly, we found that HPDMs generate forces that mechanically rupture 25 basepair DNA duplexes and biotin-streptavidin bonds (the strongest noncovalent bonds found in nature with 100+ pN force thresholds). HPDMs lack directed tracks and conformational switching such as ATP-fueled powerstrokes, thus underscoring the novelty of this result. To study this fundamental mechanism of force-generation, we developed a simulation method that accurately reproduces most properties of HPDM motion via direct modeling of the distance-dependant biophysics of DNA-RNA interactions. These simulations highlight the mechanism of HPDM force generation and demonstrate that motion is driven by autochemophoresis, which has been observed in biological systems (Sugawara & Kaneko, Biophysics 2011). Our work suggests that autochemophoresis may be a third fundamental method of force generation in molecular motors and living systems.

DNA–RNA interactions are critical for chromosome condensation in Escherichia coli

Bacterial chromosome (nucleoid) conformation dictates faithful regulation of gene transcription. The conformation is condition-dependent and is guided by several nucleoid-associated proteins (NAPs) and at least one nucleoid-associated noncoding RNA, naRNA4. Here we investigated the molecular mechanism of how naRNA4 and the major NAP, HU, acting together organize the chromosome structure by establishing multiple DNA-DNA contacts (DNA condensation). We demonstrate that naRNA4 uniquely acts by forming complexes that may not involve long stretches of DNA-RNA hybrid. Also, uncommonly, HU, a chromosome-associated protein that is essential in the DNA-RNA interactions, is not present in the final complex. Thus, HU plays a catalytic (chaperone) role in the naRNA4-mediated DNA condensation process.

A Novel Suppressive Long Noncoding RNA within the IGF1R Gene Locus Is Imprinted in Acute Myelocytic Leukemia

Background：In acute myeloid leukemia (AML) cells, the insulin-like growth factor type I receptor (IGF1R) is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth and therapeutic resistance through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in AML. Long non-coding RNAs have been implicated in a variety of important biological processes by epigenetic mechanisms.Materials and methods：AML cell lines used in this study, KG-1, KG-1a were purchased from ATCC. The full length of IRAIN lncRNA was characterized by Marathon cDNA Amplification Kit. For qPCR, cDNA samples were amplified using CFX96TM real-time system (BIO-RAD) by SYBR PrimeScript™ RT-PCR Kit (TaKaRa). Both reverse transcription-associated trap (RAT) and chromatin conformation capture (3C) assays were used to examine the chromosome architecture of IGF1R and DNA-RNA interaction. The protocol of AMLpatients bone marrow and peripheral blood cell samples was approved by the Human Medical Ethical Review Committee from Jilin University First Hospital and informed consent was obtained from each AML patient and normal donor.Results：1. we discovered a novel intragenic 5366bp long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter.2. we demonstrated IRAIN was expressed exclusively from the paternal allele, with the maternal counterpart being silenced in normal donor and AML patients.3. we confirmed that IRAIN interacts with IGF1R chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction.4. We demonstrated IRAIN was downregulated both in AML cell lines and high risk AML patients. Furthermore，the expression of IRAIN was dramatical higher in APL patients achieved molecular remission.Conclusion：Our results identify IRAIN as a new imprinted suppressive lncRNA involved in long range IGF1R DNA interactions in AML. As a putative tumor suppressor and a novel treatment target， further studies are needed to delineate the specific role of this newly identified lncRNA in the uregulation of the IGF pathway in AML. DisclosuresNo relevant conflicts of interest to declare.

A Sparse Regulatory Network of Copy-Number Driven Gene Expression Reveals Putative Breast Cancer Oncogenes

Copy number aberrations are recognized to be important in cancer as they may localize to regions harboring oncogenes or tumor suppressors. Such genomic alterations mediate phenotypic changes through their impact on expression. Both cis- and transacting alterations are important since they may help to elucidate putative cancer genes. However, amidst numerous passenger genes, trans-effects are less well studied due to the computational difficulty in detecting weak and sparse signals in the data, and yet may influence multiple genes on a global scale. We propose an integrative approach to learn a sparse interaction network of DNA copy-number regions with their downstream transcriptional targets in breast cancer. With respect to goodness of fit on both simulated and real data, the performance of sparse network inference is no worse than other state-of-the-art models but with the advantage of simultaneous feature selection and efficiency. The DNA-RNA interaction network helps to distinguish copy-number driven expression alterations from those that are copy-number independent. Further, our approach yields a quantitative copy-number dependency score, which distinguishes cis- versus trans-effects. When applied to a breast cancer data set, numerous expression profiles were impacted by cis-acting copy-number alterations, including several known oncogenes such as GRB7, ERBB2, and LSM1. Several trans-acting alterations were also identified, impacting genes such as ADAM2 and BAGE, which warrant further investigation. An R package named lol is available from www.markowetzlab.org/software/lol.html.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases.

Genomic imprinting: Parental differentiation of the genome

Genomic imprinting is the biological process whereby a gene or genomic domain exists in a state of epigenetic differentiation that depends upon its parent of origin. Importantly, the establishment and propagation of these parent-specific genomic conformations does not alter the primary DNA sequence comprised of A, C, G, and T nucleotides. Genomic imprints may be covalent (DNA methylation) or non-covalent (DNA-protein and DNA-RNA interactions, genomic localization in nuclear space), and the process of imprinting encompasses the specialized nuclear enzymatic machinery that maintains parental epigenetic markings throughout the cell cycle. Because of genomic imprinting, the parent of origin of homologous genetic alleles in diploid individuals can be determined in the absence of DNA sequence polymorphisms and without recourse to parental DNA samples. As illustrated in Figure 1, alleles of imprinted genes look and behave differently, as determined by parent of origin.

Disambiguating epigenetics

An understanding of epigenetics is now essential for the clinical pathologist in terms of disease pathogenesis, prognostication and therapeutic opportunities. Yet epigenetics, the study of inherited altered gene expression despite an unchanged DNA sequence, remains a source of confusion for many. For example, it is often difficult to appreciate parent-of-origin effects involving genomic imprinting including covalent (DNA methylation) and non-covalent changes (DNA-protein and DNA-RNA interactions, chromatin remodelling). In this talk I will highlight the importance of developments in RNA biology with a special focus on non-coding RNAs and microRNAs. Burgeoning subsets of RNA molecules are being identified that do not encode protein and function predominately as regulators of gene expression.¹ One of the most widely studied subset contains microRNAs, which are tiny endogenously-expressed molecules that fine-tune gene expression through several different mechanisms.² In the case of haematological malignancies, profiling of microRNA expression has been shown to correlate with cytogenetic changes, somatic mutations and clinical outcomes in acute myeloid leukaemia, chronic myeloid leukaemia, chronic lymphocytic leukaemia and lymphoma.

Identifying Coevolving Partners from Paralogous Gene Families

Many methods have been developed to detect coevolution from aligned sequences. However, all the existing methods require a one-to-one mapping of candidate coevolving partners (nucleotides, amino acids) a priori. When two families of sequences have distinct duplication and loss histories, finding the one-to-one mapping of coevolving partners can be computationally involved. We propose an algorithm to identify the coevolving partners from two families of sequences with distinct phylogenetic trees. The algorithm maps each gene tree to a reference species tree, and builds a joint state of sequence composition and assignments of coevolving partners for each species tree node. By applying dynamic programming on the joint states, the optimal assignments can be identified. Time complexity is quadratic to the size of the species tree, and space complexity is exponential to the maximum number of gene tree nodes mapped to the same species tree node. Analysis on both simulated data and Pfam protein domain sequences demonstrates that the paralog coevolution algorithm picks up the coevolving partners with 60% 88% accuracy. This algorithm extends phylogeny-based coevolutionary models and make them applicable to a wide range of problems such as predicting protein-protein, protein-DNA and DNA-RNA interactions of two distinct families of sequences.

Transcription termination directed by heteroduplex thr attenuator templates: Evidence that the transcript stem and loop structure is the termination signal

We have constructed both homoduplex and heteroduplex restriction fragment templates that contained mutations in the G + C-rich region of dyad symmetry in the Escherichia coli thr operon attenuator and have studied their termination properties in an in vitro transcription system. We found that the DNA sequence of the template strand determines the transcription termination efficiency. We also constructed two double mutant heteroduplex templates to examine the role of possible complementary base pairs between the nontranscribed DNA strand and the nascent RNA in the transcription bubble. There was no indication that the potential DNA-RNA interactions in the G + C-rich region are involved in transcription termination. Taken together, our results are consistent with and extend those obtained by Ryan and Chamberlin (Ryan, T., and Chamberlin, J. (1983) J. Biol. Chem. 258, 4690-4693) for the trp operon attenuator and argue strongly against models that propose that DNA hairpin formation in the nontranscribed DNA strand or pairing of the nascent RNA with the nontranscribed DNA strand are important for transcription termination.

Folded chromosome structure and DNA-binding protein of Streptomyces hygroscopicus

The isolation of the folded chromosomal structure from a Streptomyces strain is described. It is shown that the compact DNA structure of Streptomyces hygroscopicus is stabilized by DNA-RNA interactions. Nucleolytic cleavage decreases the sedimentation rate of the originally isolated (membrane-free) folded Streptomyces chromosome from 1500 S to 800 S or 300 S and finally to completely unfolded DNA. Data on ethidium bromide intercalation suggest a dye-induced relaxation and reintroduction of DNA supertwisting. Like naturally occurring covalently closed circular DNA and like the Escherichia coli chromosome, the folded chromosomal DNA of S. hygroscopicus has about one negative superhelix turn per 200 bp. The results further demonstrate that the isolated Streptomyces nucleoid contains a characteristic S protein which exhibits gel electrophoretic properties of a histone-like component similar to that found in E. coli or Thermoplasma acidophilum. Digestion of the nucleoid with proteinase K at 37°C causes elimination of the S protein and unfolding of the compact structure. The S protein is also present in other species of the genus Streptomyces.

A general method of gene isolation.

A general method of gene isolation has been developed that involves the chemical linkage of RNA to cellulose by a water-soluble carbodiimide, and the continuous circulation of DNA containing specific sequences complementary to the RNA. The temperature of the cellulose matrix is maintained at 37 degrees (50% formamide, 0.3 M NaCl-0.03 M Na(3) citrate) to allow efficient DNA-RNA interaction in the stationary phase, while unreacted and any reassociated DNA is denatured at 90 degrees and then recirculated into the hybridization chamber. Between 40 and 45% of fragmented (32)P-labeled simian virus (SV)40 DNA was removed from the circulating solution when cellulosebound SV40-specific RNA, assymmetrically transcribed in vitro with Escherichia coli RNA polymerase, was used. In the presence of 10(4)-fold excess of sheared E. coli DNA, nearly half of the [(32)P]SV40 DNA was recovered from the mixture as a DNA-RNA hybrid with negligible contamination by bacterial DNA. The isolation procedure is almost quantitative for the complementary DNA. The efficiency and selectivity of this method permit the isolation of a defined DNA sequence from a large and complex genome.