Short Synthetic DNA Research Articles

Effect of intercalator substituent and nucleotide sequence on the stability of DNA- and RNA-naphthalimide complexes

DNA intercalators are commonly used as anti-cancer and anti-tumor agents. As a result, it is imperative to understand how changes in intercalator structure affect binding affinity to DNA. Amonafide and mitonafide, two naphthalimide derivatives that are active against HeLa and KB cells in vitro, were previously shown to intercalate into DNA. Here, a systematic study was undertaken to change the 3-substituent on the aromatic intercalator 1,8-naphthalimide to determine how 11 different functional groups with a variety of physical and electronic properties affect binding of the naphthalimide to DNA and RNA duplexes of different sequence compositions and lengths. Wavelength scans, NMR titrations, and circular dichroism were used to investigate the binding mode of 1,8-naphthalimide derivatives to short synthetic DNA. Optical melting experiments were used to measure the change in melting temperature of the DNA and RNA duplexes due to intercalation, which ranged from 0 to 19.4°C. Thermal stabilities were affected by changing the substituent, and several patterns and idiosyncrasies were identified. By systematically varying the 3-substituent, the binding strength of the same derivative to various DNA and RNA duplexes was compared. The binding strength of different derivatives to the same DNA and RNA sequences was also compared. The results of these comparisons shed light on the complexities of site specificity and binding strength in DNA–intercalator complexes. For example, the consequences of adding a 5′-TpG-3′ or 5′-GpT-3′ step to a duplex is dependent on the sequence composition of the duplex. When added to a poly-AT duplex, naphthalimide binding was enhanced by 5.6–11.5°C, but when added to a poly-GC duplex, naphthalimide binding was diminished by 3.2–6.9°C.

Glucose Conjugation of Anti-HIV-1 Oligonucleotides Containing Unmethylated CpG Motifs Reduces Their Immunostimulatory Activity

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted.Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis

Attomole (10−18mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16amole detection of 1563nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the “alarm signal” range (from 1 to 100 cells per 100mL).

The importance of being modular

DNA is the material of choice for making custom-designed, nanoscale shapes and patterns through self-assembly. A new technique revisits old ideas to enable the rapid prototyping of more than 100 such DNA shapes. See Letter p.623 Programmed DNA self-assembly is widely used to create nanometre-sized structures. Modular strategies promise simplicity and versatility, yet cannot easily assemble large numbers of small strands into prescribed and complex shapes. Peng Yin and colleagues overcome this problem by designing a molecular canvas: a rectangle assembled from single-stranded tiles each consisting of a short and unique 42-base DNA strand that folds into a 3-nanometre-by-7-nanometre tile and attaches to 4 neighbouring tiles. A desired shape, drawn on the canvas, is produced by simply mixing those strands that correspond to pixels covered by the target shape, and excluding 'off'-pixel strands. With a master strand collection for a 310-pixel canvas, the team then creates more than 100 distinct and complex two-dimensional shapes that establish the method as a simple, modular and robust framework for assembling short synthetic DNA strands into complex DNA nanostructures.

Complex shapes self-assembled from single-stranded DNA tiles

Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.

Correction of Stress-induced Dysfunctions of the Immune and Neuroendocrine Systems by Peptide and Nucleotide Preparations

The authors analyzed the effects of short synthetic peptides and native DNA preparations on the function of the immune and neuroendocrine systems under various stress conditions. These preparations of peptides and nucleotides are known to be effective modulators of the immune and neuroendocrine systems. Here we discuss a new concept, which suggests that endogenous short peptides and their synthetic analogs bind to specific sequences of nucleotides in DNA strands. These site-specific peptide- DNA interactions modulate cellular genetic functions and form the basis of molecular-genetics of stress-protective short synthetic peptides. Protective action of nucleotide preparations on impaired functions of the immune and neuroendocrine systems was shown. It seems that these effects are based on the ability of nucleotides to penetrate cells and subsequently splitting into nucleotides, which, after releasing from the cell, bind to purinergic P2 receptors. The results indicate that short synthetic peptides and native DNA preparations are capable of correcting stress induced impairment of neuroimmune function.

Single-Molecule Fluorescence Meets DNA Origami

Recent developments in DNA nanotechnology facilitate a new degree of control to place arbitrary objects. Therefore, DNA is folded into arbitrary 2- and 3-dimensional structures on a nanometer to micrometer scale. In this so-called origami technique, introduced by Paul Rothemund in 2006, one ∼7.3 kbases long single-stranded DNA is hybridized with ∼200 short synthetic DNA “staple” strands to build a desired structure by self-assembly. Objects of interest, e.g. single fluorophores, are attached to individual incorporated DNA strands at specific positions within the structure. Several applications of this approach are shown using single-molecule fluorescence techniques.Revisiting the distance dependence of fluorescence resonance energy transfer (FRET), we used the DNA origami technique to build a spectroscopic ruler. In contrast to double stranded DNA, a commonly used spacer molecule, this technique offers distinct advantages. We designed a rigid DNA origami block, which has a higher persistence length and additionally allows placing the dye molecules all oriented in the same direction on the top surface, limiting static effects of the linker lengths. In contrast to dsDNA, for the origami block the Forster Radius R0 could directly be obtained from the distance dependence of energy transfer based on single-molecule FRET measurements.Guided by the idea to build complex spectroscopic networks by self-assembly, we used rectangular DNA origami as a molecular breadboard to precisely position individual fluorophores. In this artificial system the path of energy transfer can be manipulated on the nanoscale. Fluorophores were incorporated such that the light from the “blue” input dye could either be guided to the “red” or “IR” output dye, by a “green” dye that was placed at two alternative positions. We used a single-molecule four-color FRET approach with alternating laser excitation for analysis of the different energy transfer paths.

Making sense of therapeutics using antisense technology

Introduction: Antisense oligonucleotides (ASOs) are short synthetic single-stranded DNA sequences that bind to and induce the cleavage of homologous stretches of mRNA sequences. These result in targeted destruction of mRNA and correction of genetic aberrations. ASOs thus can act as drug molecules and potentially rectify many disease conditions. The broad range of applications reported in the literature highlights the advances in the field.Areas covered: This review covers different areas in which use of ASOs has been shown to have therapeutic effects. Some drugs in different stages of preclinical and clinical trials are discussed in detail. The problems faced and the strategies to surmount them are also described. The readers will gain an understanding of the recent developments in the field of ASOs with emphasis on their therapeutic applications. They will also become aware of the different strategies used for targeted delivery of ASOs and their stabilization, which may be useful for their work in this field, or in the area of nucleic acid therapeutics in general.Expert opinion: The design and application of ASOs for recognition of target mRNA sequences have become a fairly straightforward protocol. The main problem lies in designing ASOs which are stable in in vivo milieu. The delivery and bioavailability of the oligonucleotide to the site of action continue to be hurdles in the development of ASOs and therapeutic molecules.

Influence of percentage of guanine molecules, OH radicals, UV irradiation and temperature on electrooxidation of short synthetic oligonucleotides

The electrooxidation of short synthetic 20-nucleotides DNA sequences with various amount of guanine molecules has been studied in a wide temperature range by square wave voltammetry and the results were compared with UV-vis and CD spectra. A twofold increase of dsDNA voltammetric peak, related to an increase in the number of electrons transferred in the guanine electrooxidation process was found to begin at a temperature lower by circa 20 °C compared to the well known increase of the dsDNA absorbance upon denaturation. Since the dsDNA voltammetric peaks are related directly to the electrooxidation of guanine and adenine, early conformational changes in dsDNA are responsible for this effect. An increase in percentage of guanine in the DNA chains caused a delay in the conformational, predenaturation changes. An exception to this behavior was found for polyguanine (100% guanine). Interestingly, two distinct ranges of change in ellipticity in the CD spectra correlate well with the changes obtained by voltammetry. We have also checked the influence of OH radicals and UV irradiation on the dsDNA oxidation.

Bottlenecks and the Maintenance of Minor Genotypes during the Life Cycle of Trypanosoma brucei

African trypanosomes are digenetic parasites that undergo part of their developmental cycle in mammals and part in tsetse flies. We established a novel technique to monitor the population dynamics of Trypanosoma brucei throughout its life cycle while minimising the confounding factors of strain differences or variation in fitness. Clones derived from a single trypanosome were tagged with short synthetic DNA sequences in a non-transcribed region of the genome. Infections were initiated with mixtures of tagged parasites and a combination of polymerase chain reaction and deep sequencing were used to monitor the composition of populations throughout the life cycle. This revealed that a minimum of several hundred parasites survived transmission from a tsetse fly to a mouse, or vice versa, and contributed to the infection in the new host. In contrast, the parasites experienced a pronounced bottleneck during differentiation and migration from the midgut to the salivary glands of tsetse. In two cases a single tag accounted for ≥99% of the population in the glands, although minor tags could be also detected. Minor tags were transmitted to mice together with the dominant tag(s), persisted during a chronic infection, and survived transmission to a new insect host. An important outcome of the bottleneck within the tsetse is that rare variants can be amplified in individual flies and disseminated by them. This is compatible with the epidemic population structure of T. brucei, in which clonal expansion of a few genotypes in a region occurs against a background of frequent recombination between strains.

Multilayer DNA Origami Packed on a Square Lattice

Molecular self-assembly using DNA as a structural building block has proven to be an efficient route to the construction of nanoscale objects and arrays of increasing complexity. Using the remarkable "scaffolded DNA origami" strategy, Rothemund demonstrated that a long single-stranded DNA from a viral genome (M13) can be folded into a variety of custom two-dimensional (2D) shapes using hundreds of short synthetic DNA molecules as staple strands. More recently, we generalized a strategy to build custom-shaped, three-dimensional (3D) objects formed as pleated layers of helices constrained to a honeycomb lattice, with precisely controlled dimensions ranging from 10 to 100 nm. Here we describe a more compact design for 3D origami, with layers of helices packed on a square lattice, that can be folded successfully into structures of designed dimensions in a one-step annealing process, despite the increased density of DNA helices. A square lattice provides a more natural framework for designing rectangular structures, the option for a more densely packed architecture, and the ability to create surfaces that are more flat than is possible with the honeycomb lattice. Thus enabling the design and construction of custom 3D shapes from helices packed on a square lattice provides a general foundational advance for increasing the versatility and scope of DNA nanotechnology.

Rapid Oligonucleotide-Based Recombineering of the Chromosome of Salmonella enterica

Recombinant engineering using Red recombinase-based approaches offers efficient and rapid approaches to deletion and modification of genes. Here we describe a novel application of Red recombinant engineering that enables direct manipulation of chromosomal loci by electroporation with short synthetic DNA molecules. We demonstrate the use of this approach for the generation of scarless in-frame deletions in chromosomal genes of Salmonella enterica. Furthermore, we describe rapid site-directed mutagenesis within bacterial chromosomes without any requirement for cloning and mutating genes in vitro or for reintroducing mutant alleles into the chromosome. This approach can be expected to facilitate mutational analysis in S. enterica and in other bacterial species able to support Red-mediated recombination.

Biophysical Studies on the Stability of DNA Intrastrand Cross-Links of Transplatin

Clinically ineffective transplatin [ trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.

Creation of Mammalian Single- and Double-Stranded DNA Surfaces: A Real-Time QCM-D Study

The quartz crystal microbalance with dissipation monitoring (QCM-D) is an excellent method for studying the creation of DNA-based surfaces and films. Previous studies have used QCM-D to focus on the construction of DNA surfaces composed of short synthetic DNA oligomers or plasmid DNA. Here, we have used QCM-D to monitor the creation of genomic single- and double-stranded calf thymus DNA surfaces on a polycation adsorbed to a SiO2 support. We have successfully monitored the hybridization between the ssDNA surfaces and their complementary strands in solution and have also shown that DNA multilayer formation can be observed using denatured calf thymus DNA. We have furthermore established that the ssDNA and dsDNA surfaces show different binding characteristics to ethidium bromide, a common dsDNA intercalator, demonstrating the potential use of such surfaces to identify possible DNA ligands.

Characterisation of the novel restriction endonuclease SuiI from Sulfolobus islandicus

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70 degrees C.

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.

The Future of Antisense Oligonucleotides in the Treatment of Respiratory Diseases

Antisense oligonucleotides (ASO) are short synthetic DNA molecules designed to inhibit translation of a targeted gene to protein via interaction with messenger RNA. More recently, small interfering (si)RNA have been developed as potent tools to specifically inhibit gene expression. ASO directed against signaling molecules, cytokine receptors, and transcription factors involved in allergic immune and inflammatory responses, have been applied in experimental models of asthma and demonstrate potential as therapeutics. Several ASO-based drugs directed against oncogenes have been developed for therapy of lung cancer, and some have recently reached clinical trials. ASO and siRNA to respiratory syncytial virus infection have demonstrated good potential to treat this condition, particularly in combination with an antiviral drug. Although ASO-based therapeutics are promising for lung diseases, issues of specificity, identification of correct molecular targets, delivery and carrier systems, as well as potential adverse effects must be carefully evaluated before clinical application.

Quenched probes for highly specific detection of cellular RNAs

Nucleic acid-based RNA detection is a promising field in molecular biotechnology that is leading to the rapid and accurate identification of microorganisms, diagnosis of infections and imaging of gene expression. The specificity of short synthetic DNA probes raises the hope of distinguishing small differences in sequence, ultimately achieving single nucleotide resolution. Recent work using quenched fluorescently labeled oligonucleotide probes as sensors for RNA in bacterial and human cells has overcome several difficult hurdles on the way to these goals, including delivery of probes to live cells, accessing RNA sites containing a high degree of secondary structure, and eliminating many sources of background. Two new classes of quenched oligonucleotide probes, molecular beacons and quenched auto-ligation probes, have shown the most promise for in situ RNA detection. High-specificity detection, at the single-nucleotide resolution level, is now possible in solution with these classes of probes. However, for applications in intact cells, signal and background issues still need to be addressed before the full potential of these methods is achieved.

Validating siRNA using a reporter made from synthetic DNA oligonucleotides

Only a small fraction of all siRNAs are effective in silencing their target genes, and siRNA efficacy can only be determined experimentally. Previously described reporter-based siRNA validation methods all rely on the availability of physical cDNA clones, and this limits the high throughput applicability of the method. In the current report, we used short synthetic DNA fragment containing a siRNA targeting site, instead of cDNA, to fuse with a reporter gene. When targeting such transcripts with different siRNAs, we found that such constructs can faithfully report the efficacy of the corresponding siRNAs in a sequence specific manner, even when the inserted DNA fragment is essentially only long enough to cover the targeting site. The efficacy of both vector-based siRNA and synthetic siRNA can be evaluated using this system. Since only readily available short synthetic DNA fragments are needed for forming the evaluation vector, this method provides an appealing way of validating siRNAs in high throughput.