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Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3′UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.

Novel 4-aminoantipyrine based mixed ligand metal complexes with the Schiff bases ofL¹(L¹-4(furanylmethyleneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one and L²/ L³/ L⁴are 2-(2-chlorobenzylideneamino)phenol, 2-(3-chlorobenzylideneamino)phenol, 2-(4-chlorobenzylideneamino)phenol were synthesized. The structures of the mixed ligand complexes were established by analytical and spectral techniques. They were screened for in vitro antimicrobial activity against bacteria and fungi by disc diffusion method. The interaction of metal complexes with CT-DNA was investigated by UV–vis, cyclic voltammetry, viscosity and thermal denaturation studies.DNA interaction studies suggest that metal complex binds to calf thymus DNA (CT-DNA) through intercalation mode. Superoxide dismutase activity of these complexes has also been studied. The free ligands and their metal complexes have been tested for in vitro antioxidant activity by the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH).The antioxidant activities of the complexes were studied and compared with the activity of ascorbic acid. Cu(II) complex showed superior antioxidant activity than other complexes. The solvatochromic behaviour of complexes was also performed in various solvents.

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5'-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5'-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization. We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes. Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA. This study defines how a short DNA sequence, known as the PAM, is critical for target DNA interrogation by the CRISPR-associated enzyme Cas9 — DNA melting and heteroduplex formation initiate near the PAM and extend directionally through the remaining target sequence, and the PAM is also required to activate the catalytic activity of Cas9. Role of PAM in CRISPR/Cas9 genome editing CRISPR/Cas9-based DNA targeting has quickly become a leading tool in the fields of synthetic biology and genome engineering. It exploits the ability of a bacterial endonuclease, Cas9, guided by an RNA molecule, to target virtually any matching DNA sequence of interest for binding and/or cleavage. This paper reports the use of single-molecule and bulk biochemical experiments to reveal the mechanism by which RNA-guided Cas9 locates unique 20-base-pair sequences within DNA genomes, which can be billions of base pairs long. The results highlight the role of a trinucleotide protospacer adjacent motif (PAM) in recruiting Cas9–RNA complexes to potential DNA target sites, and in catalytically activating the nuclease. Target DNA sequences are recognized via a 'zip-up' mechanism, where the sequential formation of RNA–DNA base pairs offsets the energetic cost of unwinding the DNA double helix. In addition to its relevance for gene manipulation, this work reveals how DNA is interrogated by Cas9–RNA in its role as an effector of adaptive immunity in bacteria.

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than most polymerases can exert 1 , 2 . Eukaryotic cells contain numerous helicases that can unfold G-quadruplexes 3 . The molecular basis of the recognition and unfolding of G-quadruplexes by helicases remains poorly understood. DHX36 (also known as RHAU and G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with extremely high affinity 4 – 6 , is consistently found bound to G-quadruplexes in cells 7 , 8 , and is a major source of G-quadruplex unfolding activity in HeLa cell lysates 6 . DHX36 is a multi-functional helicase that has been implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, haematopoiesis, and embryogenesis in mice 9 – 12 . Here we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3′ single-stranded DNA segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that, together with the OB-fold-like subdomain, selectively binds parallel G-quadruplexes. Comparison with unliganded and ATP-analogue-bound DHX36 structures, together with single-molecule fluorescence resonance energy transfer (FRET) analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core; by pulling on the single-stranded DNA tail, these rearrangements drive G-quadruplex unfolding one residue at a time. A mechanism for the unfolding of guanine-rich DNA ‘quadruplexes’ by helicases is suggested, based on the structure of a DNA-bound helicase.

Abstract Melting and fast cooling double stranded DNA donor molecules prior to injection dramatically increases the frequency of homology-directed repair for edits such as insertions of fluorescent protein markers in Caenorhabditis elegans. Strategies described here enable consistently ... Abstract CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans, one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring coselection strategies. Here, we show that melting double-stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

Understanding how RNA structure influences its function has been hampered by a lack of approaches that can accurately quantify RNA structure in vivo ; here, RNA structure is revealed on a global scale and with nucleotide-level resolution, showing that there is less structure within cells than expected from in vitro and in silico analyses. Probing the in vivo RNA structurome Being single-stranded, RNA can adopt a diversity of secondary structures via inter- and intramolecular base-pairing. Three studies published in this issue of Nature provide an in-depth view of the variety, dynamics and functional influence of RNA structures in vivo . Sarah Assmann and colleagues map the in vivo RNA structure of over 10,000 transcripts in the model plant Arabidopsis thaliana . Their struc-seq (structure-seqence) approach incorporates in vivo chemical (DMS) probing and next-generation sequencing to provide single-nucleotide resolution on a genome-wide scale. Distinct patterns of structure are found to be correlated with coding regions, splice sites and polyadenylation sites. Comparison of these results with those obtained by earlier technologies reveals that, although predictions for some classes of genes were fairly accurate, others, such as those involved in stress response, were poorly predicted and may reflect changes that made them more adapted to that condition. Jonathan Weissman and colleagues have also developed a DMS-seq method to globally monitor RNA structure to single-nucleotide precision in yeast and mammalian cells. Comparing their findings with in vitro data, the authors conclude that there is less structure within cells than expected. Even thermostable RNA structures can be denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Howard Chang and colleagues asked a different question: how does RNA secondary structure change on a transcriptome-wide level in related individuals? By calculating the RNA secondary structures of two parents and their child, they find that about 15% of transcribed single-nucleotide variants affect local secondary structure. These 'RiboSNitches' are depleted in certain locations, suggesting that a particular RNA structure at that site is important. This study illustrates that there is much to be learned about how changes in RNA structure, particularly as imparted by genetic variation, can alter gene expression. RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA’s ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational 1 , 2 and experimental 3 , 4 , 5 , 6 , 7 , 8 efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo 5 . Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues 9 , followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome 10 . Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro . Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo .

Acridines and their derivatives are well-known probes for nucleic acids as well as being relevant in the field of drug development to establish new chemotherapeutic agents. We have shown from molecular modelling studies that 9-phenyl acridine and some of its derivatives can act as inhibitors of topoisomerase I and thus have potential to act as anticancer agents. Rational design of new compounds for therapeutics requires knowledge about their structural stability and interactions with various cellular macromolecules. In this regard it is important to know how these molecules would interact with DNA. Here we report the interaction of 9-phenyl acridine (ACPH) with calf thymus DNA (CT-DNA) based on various biophysical and molecular modelling studies. Spectrophotometric studies indicated that ACPH binds to CT-DNA. DNA melting studies revealed that binding of ACPH to CT-DNA resulted in a small increase in melting temperature, which is unlikely in case of classical intercalator; rather, it indicates external binding. Viscosity measurements show that ACPH exhibits groove binding. Competitive binding of ACPH to CT-DNA pre-bound to ethidium bromide (EB) showed slow quenching. Measurement of the binding constant of ACPH by fluorescent intercalator displacement (FID) assay corroborated the notion that there was groove binding. Molecular modelling studies also supported this finding. Results indicate that binding of ACPH is through partial intercalation in the minor groove of DNA.

Hereditary breast cancer accounts for about 10% of all breast cancers and BRCA1 and BRCA2 genes have been identified as validated susceptibility genes for this pathology. Testing for BRCA gene mutations is usually based on a pre-screening approach, such as the partial denaturation DHPLC method, and capillary direct sequencing. However, this approach is time consuming due to the large size of BRCA1 and BRCA2 genes. Recently, a new low cost and time saving DHPLC protocol has been developed to analyze gene mutations by using SURVEYOR® Nuclease digestion and DHPLC analysis. A subset of 90 patients, enrolled in the Genetic Counseling Program of the National Cancer Centre of Bari (Italy), was performed to validate this approach. Previous retrospective analysis showed that 9/90 patients (10%) were mutated in BRCA1 and BRCA2 genes and these data were confirmed by the present approach. DNA samples underwent touchdown PCR and, subsequently, SURVEYOR® nuclease digestion. BRCA1 and BRCA2 amplicons were divided into groups depending on amplicon size to allow multiamplicon digestion. The product of this reaction were analyzed on Transgenomic WAVE Nucleic Acid High Sensitivity Fragment Analysis System. The operator who performed the DHPLC surveyor approach did not know the sequencing results at that time. The SURVEYOR® Nuclease DHPLC approach was able to detect all alterations with a sensitivity of 95%. Furthermore, in order to save time and reagents, a multiamplicon setting preparation was validated.

Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposi's sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3′-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3′-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique.