Small Oligonucleotides Research Articles

3′-Amino-5′-carboxymethyl-3′,5′-dideoxy nucleosides for the synthesis of fully amide-linked RNA mimics

A convenient protocol is developed for the synthesis of 3′-[N-(fluorenylmethoxycarbonyl)-amino]-5′-carboxymethyl derivatives of all four natural ribonucleosides from cheap chiral pool compound glucose. Synthesis of fully amide-linked RNA analogues of small oligonucleotides containing, for the first time, all four nucleoside amino acids using standard solid phase Fmoc-chemistry is described.

Functional nucleic-acid-based sensors for environmental monitoring.

Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and non-immunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleic-acid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental monitoring.

Enhancing siRNA delivery by employing lipid nanoparticles.

For several decades extensive research has been conducted into the development of fusogenic lipid nanoparticles (LNPs) capable of introducing large, charged molecules into the cytoplasm of target cells. The majority of this work has focused on cationic LNPs encapsulating nucleic acids ranging from small oligonucleotides to large plasmid constructs thousands of bases long. However, since the introduction of siRNA payloads this quest for a non-viral, intracellular delivery systems has advanced significantly. Of particular importance was the demonstration that LNPs containing ionizable, dialkylamino lipids, enable potent hepatic gene silencing across species including humans. This review focuses on the evolution of this delivery system, summarizes the promising data now emerging from clinical trials and considers future directions for the platform.

Detection of microRNA in tumor cells using exonuclease III and graphene oxide-regulated signal amplification.

In this study, we developed a label-free, ultrasensitive graphene oxide (GO)-based probe for the detection of oligonucleotides by laser desorption/ionization mass spectrometry (LDI-MS). On the basis of simple π-π stacking and electrostatic interactions between rhodamine 6G (R6G) and GO, we prepared the nanocomposite R6G-modified GO (R6G-GO). Signal intensities of R6G increased in mass spectra in the presence of single-stranded oligonucleotides under pulsed laser irradiation (355 nm) of R6G-GO. In addition, the signal intensity of R6G was stronger in the presence of short oligonucleotides. Because small oligonucleotides improve the LDI efficiency of R6G on GO, we designed an enzyme-amplified signal transduction probe system for the detection of microRNA (miRNA). After specific digestion of the probe DNA (pDNA) strand from pDNA/miRNA-hybridized complexes by exonuclease III (Exo III), the resulting small oligonucleotide fragments increased the R6G signal during LDI-MS of R6G-GO. In addition, the signal intensity of the R6G ions increased with increasing concentrations of the target miRNA. Coupling this enzyme reaction and R6G-GO with LDI-MS enabled the detection of miRNA at concentrations of the femtomolar (fM) level. We also demonstrated the analysis of miRNA in tumor cells and utilized this R6G-GO probe in the detection of a single-nucleotide polymorphism (SNP) in the Arg249Ser unit of the TP53 gene. This simple, rapid, and sensitive detection system based on the coupling of functional GO with LDI-MS appears to have great potential as a tool for the bioanalyses of oligonucleotides and proteins.

Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m (99mTc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with 99mTc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA−), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with 99mTc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.

Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma.

The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.

Structural and Functional Studies of a Novel RNA-Binding Sm-Like Archaeal Protein

In all three domains of life, Sm oligomers interact with RNA, either as chaperones or as scaffolding to facilitate RNA···RNA and RNA···protein interactions. In bacteria and eukarya, Sm homologs are key for RNA metabolism, for instance in RNA-mediated post-transcriptional regulation in bacteria. However, the in vivo roles of Sm-like archaeal proteins (SmAPs) remain unknown. Sequence searches suggest that the hyperthermophilic crenarchaeote Pyrobaculum aerophilum (Pae) encodes three SmAPs. The two known Pae Sm structures illuminated Sm protein evolution and assembly, and implied that these homologs may represent an ancestral form of the heteromeric Sm complexes that pervade eukaryotic RNA processing, e.g. in the spliceosome and other RNPs. We have now characterized Pae SmAP2, the final putative Sm paralog from this species. Biophysical characterization suggests that this SmAP2 exists in multiple stable oligomeric states, which we also detect via chemical cross-linking and mass spectrometry. We have now determined the crystal structure of this SmAP in multiple space-groups, revealing unexpected and hitherto unseen Sm assembly states. Binding assays with various small RNA oligonucleotides show this SmAP to be able to bind U-rich RNA, but not adenine- or cytosine-rich RNAs.

De-repressing LncRNA-Targeted Genes to Upregulate Gene Expression: Focus on Small Molecule Therapeutics.

Non-protein coding RNAs (ncRNAs) make up the overwhelming majority of transcripts in the genome and have recently gained attention for their complex regulatory role in cells, including the regulation of protein-coding genes. Furthermore, ncRNAs play an important role in normal development and their expression levels are dysregulated in several diseases. Recently, several long noncoding RNAs (lncRNAs) have been shown to alter the epigenetic status of genomic loci and suppress the expression of target genes. This review will present examples of such a mechanism and focus on the potential to target lncRNAs for achieving therapeutic gene upregulation by de-repressing genes that are epigenetically silenced in various diseases. Finally, the potential to target lncRNAs, through their interactions with epigenetic enzymes, using various tools, such as small molecules, viral vectors and antisense oligonucleotides, will be discussed. We suggest that small molecule modulators of a novel class of drug targets, lncRNA-protein interactions, have great potential to treat some cancers, cardiovascular disease, and neurological disorders.

Targeting mRNA for Alzheimer's and related dementias.

Brain deposition of the amyloid beta-protein (Aβ) and tau are characteristic features in Alzheimer's disease (AD). Mutations in the Aβ precursor protein (APP) and a protease involved in Aβ production from APP strongly argue for a pathogenic role of Aβ in AD, while mutations in tau are associated with related disorders collectively called frontotemporal lobar degeneration (FTLD). Despite intense effort, therapeutic strategies that target Aβ or tau have not yet yielded medications, suggesting that alternative approaches should be pursued. In recent years, our laboratory has studied the role of mRNA in AD and FTLD, specifically those encoding tau and the Aβ-producing protease BACE1. As many FTLD-causing tau mutations destabilize a hairpin structure that regulates RNA splicing, we have targeted this structure with small molecules, antisense oligonucleotides, and small molecule-antisense conjugates. We have also discovered that microRNA interaction with the 3′-untranslated region of tau regulates tau expression. Regarding BACE1, we found that alternative splicing leads to inactive splice isoforms and antisense oligonucleotides shift splicing toward these inactive isoforms to decrease Aβ production. In addition, a G-quadruplex structure in the BACE1 mRNA plays a role in splice regulation. The prospects for targeting tau and BACE1 mRNAs as therapeutic strategies will be discussed.

Development and Characterization of a ssDNA-based Aptamer that Selectively Targets Epithelial Carcinoma Cells

Epithelial cellular adhesion molecule (EpCAM) is a biomarker highly expressed on the surface of epithelial carcinoma cells. Consequently, it has been widely used for detection of carcinoma tumors and isolation of individual circulating cancer cells and EpCAM-targeting therapeutics are undergoing pre-clinical and clinical testing against several types of carcinomas. Aptamers are a new class of small oligonucleotide ligands that bind to their targets with high affinity and specificity, and are thus termed “chemical antibodies.” In comparison to protein antibodies, aptamers are smaller in size, which improves their tissue penetration potential, and elicit few to no immunogenic responses. Furthermore, our previous studies revealed that ssDNA, compared to RNA aptamers are significantly more stable in human serum and thus, are suitable for in vivo use. In this study, by using a hybrid cell-biomarker selection approach, we developed ssDNA-based aptamers specific for EpCAM. We identified a dominant aptamer sequence (58.7% of >0.5 million sequence reads) that contained both loop and stem structures, and specifically targeted EpCAM-expressing carcinoma cells with high binding affinity (Kd=12 nM). Validation studies indicated that the newly synthesized aptamer targeted a variety of cultured carcinoma cells, including pancreatic, breast, ovarian, prostate, and colon cancer cells, but did not react with cultured EpCAM-negative leukemia and lymphoma cells. In addition, the aptamer recognized EpCAM-expressing, patient-derived primary normal and carcinoma cells. Finally, the aptamer selectively targeted carcinoma cells in complex cell mixtures, and showed no reactivity to mononucleated or red blood cells, demonstrating its suitability for therapeutic in vivo use.

Comparing ion‐pairing reagents and sample dissolution solvents for ion‐pairing reversed‐phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides

A sensitive and selective liquid chromatography/mass spectrometry (LC/MS) method is essential for quality control of synthetic oligonucleotides. However, researchers are still searching for improvements to ion-pairing reagents for ion-pairing reversed-phase LC/MS. This study performed a comprehensive comparison of six ion-pairing reagents to determine their performance as mobile phase modifiers for oligonucleotide LC/MS. The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine) and hexafluoro-2-propanol were compared by measuring the adduct ion formation, chromatographic separation, and MS signal intensity of four oligonucleotides (10mer to 40mer). The effect of dissolution solvents on MS signal intensity and adduct ion formation was also investigated. Results showed that the type of dissolution solvent can have a signficiant impact on adduct ion formation with oligonucleotides. Results also showed that the maximum separation for small, medium and large oligonucleotides occured when using tripropylamine, N,N-dimethylbutylamine, and dibutylamine, respectively. However, on average 15 mM hexylamine and 50 mM hexafluoro-2-propanol provided the best chromtatographic performance (resolution values: 14.1 ± 0.34, 11.0 ± 0.17, and 6.4 ± 0.11 for the pairs of oligonucleotides T10 & T15, T15 & T25, and T25 & T40, respectively (3 replicates)). The impact of dissolution solvent on the MS signal of oligonucleotides depends on the type of ion-pairing reagent. Buffer combining 15 mM hexylamine and 50 mM hexafluoro-2-propanol produced the highest overall performance for oligonucleotides (10mer to 40mer) with respect to chromatographic resolution and mass detection.

STAT inhibitors for cancer therapy.

Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds.

Advances in pathogen-associated molecules detection using Aptamer based biosensors

Pathogens are all around us in the environment and although advances in medical science have been made to protect against infection by pathogens, through the use of antibiotics, vaccination and fungicide, yet pathogens continue to threaten human life. The fast pathogen detection still remains an unresolved issue since conventional identification and detection methods are complex, costly and require minimum two to three days for detection. Thus there is an urgent need for the development of robust, sensitive and portable diagnostic tools that can lead to accurate and rapid detection of a variety of pathogens. Aptamers are small DNA and RNA oligonucleotides that can bind with high affinity and selectivity to a large number of targets like small and macromolecules from organic, inorganic and biological origin. Aptasensors are biosensors where aptamers are used in place of biological ligands to sense targeted analytes. Oligonucleotide-(both DNA, RNA), peptide- and peptidenucleic acid (PNA)-based aptasensors offer high reproducibility against a wide variety of targets (proteins, peptides, drugs, metabolites, metal ions, cells etc.) and are emerging as the most suitable candidates for analytical methods that use a very small amount (nano to microlitre) of analytes. Herein, the recent developments of aptasensors for the detection of pathogens have been discussed briefly.

Interference Of The Tumor Supportive Effects Of BCL2 and MCL1 Sensitize Malignant Plasma Cells To The Lethal Effects Of Lenalidomide and Dexamethasone Regimen: An Important Clinical Path For BCL2 Targeting Drugs

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy in the U.S. and is characterized by increased immunoglobulin production and infiltration of the bone marrow by malignant plasma cells. Myeloma cell growth is supported by both the elements present in the bone marrow microenvironment as well as deregulation of internal cellular systems associated with proliferation and apoptosis. Defective programmed cell death by BCL2 or MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Various strategies to mitigate the effects of increased BCL2 functionality, via small molecule inhibitors or BCL2-specific antisense oligonucleotides have been previously examined in MM. These studies revealed that 1) apart from BCL2, additional anti-apoptotic members belonging to the BCL2 family (i.e. MCL1, BCL-xL) play integral roles in maintenance of the myeloma cell phenotype, 2) drugs, which target only BCL2 offer limited therapeutic advantage and 3) given that survival factors engaged in MM are multifactorial, perhaps isolated targeting of the BCL2 pathways may not be sufficient. We therefore hypothesized that the clinical development of drugs that target various antiapoptotic BCL2 family members will require a partnership with established anti-MM regimens whose activity can potentially be further enhanced with correction of the apoptotic response system. AimSince lenalidomide and dexamethasone (LD) is an established therapy for plasma cell cancers, we investigated if the pan-BCL2 inhibitor AT-101 (BH3 mimetic), which has high binding specificity for BCL2, MCL1 and BCL-xL, can be an effective therapeutic partner to enhance anti-MM effects of LD. MethodsHuman MM (KMS11, U266, OPM2) and WM (BCWM.1) cell lines along with their corresponding bortezomib resistant (BR) clones, (KMS11/BR, U266/BR, OPM2/BR and BCWM.1/BR) which were developed in our laboratory were used in this study. For gene expression profiling, the Illumina HumanHT-12 v3 whole-genome gene expression array and Nanostring nCounter mRNA quantification assays (NanoString, Seattle, WA) were utilized. Statistical analysis was conducted using R-based packages and the MeV software (TIGR). Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. ResultsGene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of various BCL2 family members, notably MCL1 and BCL-xL. In a dose-dependent manner, AT-101 was able to downregulate BCL2 and MCL1 and induced apoptotic cell death in MM and WM cells in a dose dependent manner. Tumor cell death was associated with caspase and PARP-1 cleavage accompanied by an increase in MOMP. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). Nanostring nCounter mRNA quantification and IPA analysis revealed differential changes in the CCNA, FRZB, FYN, IRF1, PTPN11 genes in LDA treated cells. IPA canonical pathway analysis demonstrated the p53 signaling and the cyclins and cell cycle regulation pathways to be the most significantly activated by LDA therapy. SummaryIn summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. This study lays the rationale for engaging and handicapping the intrinsic apoptotic system through the pan-BCL2 targeting capabilities of AT-101; thus enhancing the anti-MM effects of lenalidomide/dexamethasone. Disclosures:Foran:Celgene: Research Funding.

Recent Advances in Target Characterization and Identification by Photoaffinity Probes

Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL). Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs) are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes.

Manipulating the apoptotic pathway: potential therapeutics for cancer patients.

This review summarizes the current state of scientific understanding of the apoptosis pathway, with a focus on the proteins involved in the pathway, their interactions and functions. This forms the rationale for detailing the preclinical and clinical pharmacology of drugs that modulate the pivotal proteins in this pathway, with emphasis on drugs that are furthest advanced in clinical development as anticancer agents. There is a focus on describing drugs that modulate three of the most promising targets in the apoptosis pathway, namely antibodies that bind and activate the death receptors, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins, and small molecules and antisense oligonucleotides that inactivate the inhibitors of apoptosis, all of which drive the equilibrium of the apoptotic pathway towards apoptosis. These structurally different yet functionally related groups of drugs represent a promising novel approach to anticancer therapeutics whether used as monotherapy or in combination with either classical cytotoxic or other molecularly targeted anticancer agents.

Oligonucleotide Adsorption Affects Phase Transition but Not Interdigitation of diC14-Amidine Bilayers

In this work, we investigate the effect of a small single-stranded oligonucleotide (ODN) on the colloid stability and structure of cationic diC14-amidine liposomes. Dynamic light scattering (DLS) shows that small, stable, anionic assemblies are formed in presence of excess ODN negative charge. This charge overcompensation condition was further characterized. A less cooperative bilayer phase transition is observed by differential scanning calorimetry (DSC). Electron spin resonance (ESR) spectra of probes at different bilayer depths show that ODN electrostatic adsorption increases the rigidity of both interdigitated gel and lamellar fluid phases. The increase in gel phase rigidity could be explained by the transformation of an adjacent to an interpenetrated interdigitation. Interdigitated fusogenic bilayers may find interesting applications in delivery of therapeutic oligonucleotides.

Bioportides: Bioactive cell‐penetrating peptides that modulate cellular dynamics

The study and exploitation of cell-penetrating peptides (CPPs) now extends into a third exciting decade. Pharmacokinetic modulators, including the more common sequences Tat, penetratin and transportan-10, markedly enhance the intracellular delivery of small drugs, peptides, oligonucleotides and proteins. We introduced the term bioportide to distinguish cell penetrant peptides with intrinsic bioactivities from more typically inert CPP vectors. Our first examples included rhegnylogically organised bioportides, monomeric peptides presenting pharmacophores for both cellular internalization and bioactivity discontinuously distributed within the primary sequence. However, it is conceptually expedient to employ the same terminology to encompass sychnologic bioportides that comprise an inert CPP vector conjugated to an otherwise impermeable bioactive peptide. In such cases the CPP provides an obvious address function whilst the bioactive cargo, often a protein mimetic sequence, is the message. Additional targeting sequences, usually added as chimeric extensions, can also be accommodated within the design of CPPs and bioportides to enable cell- and tissue-selective targeting. Thus, the identification and exploitation of bioportides provides further scope to employ CPPs as research tools, diagnostics and therapeutics spanning a range of pathologies.

Spatial Waves in Synthetic Biochemical Networks

We report the experimental observation of traveling concentration waves and spirals in a chemical reaction network built from the bottom up. The mechanism of the network is an oscillator of the predator-prey type, and this is the first time that predator-prey waves have been observed in the laboratory. The molecular encoding of the nonequilibrium behavior relies on small DNA oligonucleotides that enforce the network connectivity and three purified enzymes that control the reactivity. Wave velocities in the range 80-400 μm min(-1) were measured. A reaction-diffusion model in quantitative agreement with the experiments is proposed. Three fundamental parameters are easy to tune in nucleic acid reaction networks: the topology of the network, the rate constants of the individual reactions, and the diffusion coefficients of the individual species. For this reason, we expect such networks to bring unprecedented opportunities for assaying the principles of spatiotemporal order formation in chemistry.