Degradation Of Oligonucleotides Research Articles

Graphene oxide-functionalized molecular beacon for real-time interference-free detection of Ki-67 mRNA in living cells

Molecular beacons (MBs) based on hairpin-shaped oligonucleotides are captivating owing to their capability to enable effective real-time detection of cytosolic mRNA in living cells. However, DNase in the nucleus and lysosome could induce the degradation of oligonucleotides in MBs, leading to the generation of false-positive signals. Herein, a graphene oxide (GO) nanosheet was applied as a nanocarrier for MBs to greatly enhance the anti-interference of the easily designed nanoprobe. Advantageously, the absorption capacity of GO for MBs increased with the decrease in pH values, providing the MB-GO nanoprobe with the ability to detect the expression of cytosolic Ki-67 mRNA without interference from DNase Ⅱ in lysosomes. Moreover, the size of GO nanosheets was considerably higher than that of the nuclear pore complex (NPC), which prevented nanoprobes from transition through the NPCs, thereby avoiding the generation of false-positive signals in the nucleus. Altogether, the present work affords a convenient approach for the successful detection of Ki-67 mRNA expression in the cytosol without interference from DNase Ⅰ/Ⅱ in the nucleus/lysosome, which may be potentially further applied for the detection of other cytosolic RNAs.

Self-Assembled Monolayers Derived from Positively Charged Adsorbates on Plasmonic Substrates for MicroRNA Delivery: A Review

MicroRNA (miRNA) has emerged as a promising alternative therapeutic treatment for cancer, but its delivery has been hindered by low cellular uptake and degradation during circulation. In this review, we discuss the various methods of delivering miRNA, including viral and non-viral delivery systems such as liposomes and nanoparticles. We also examine the use of nanoparticles for miRNA-based diagnostics. We focus specifically on non-viral delivery systems utilizing coinage metals in the form of nanoparticles and the use of self-assembled monolayers (SAMs) as a method of surface modification. We review the use of SAMs for the conjugation and delivery of small noncoding ribonucleic acid (ncRNA), particularly SAMs derived from positively charged adsorbates to generate charged surfaces that can interact electrostatically with negatively charged miRNA. We also discuss the effects of the cellular uptake of gold and other plasmonic nanoparticles, as well as the challenges associated with the degradation of oligonucleotides. Our review highlights the potential of SAM-based systems as versatile and robust tools for delivering miRNA and other RNAs in vitro and in vivo and the need for further research to address the challenges associated with miRNA delivery and diagnostics.

Detailed cellular assessment of albumin-bound oligonucleotides: Increased stability and lower non-specific cell uptake

Conjugation of lipid moieties to nucleic-acid therapeutics increases their interaction with cellular membranes, enhances their uptake and influences in vivo distribution. Once injected in biological fluids, such modifications trigger the binding of various serum proteins, which in turn play a major role in determining the fate of oligonucleotides. Yet, the role played by each of these proteins, more than 300 in serum, remains to be elucidated. Albumin, the most abundant circulating protein is an attractive candidate to study, as it was previously used to enhance the therapeutic effect of various drugs. Herein, we present a thorough fluorescent-based methodology to study the effect of strong and specific albumin-binding on the fate and cellular uptake of DNA oligonucleotides. We synthesized a library of molecules that exhibit non-covalent binding to albumin, with affinities ranging from high (nanomolar) to none. Our results revealed that strong albumin binding can be used as a strategy to reduce degradation of oligonucleotides in physiological conditions caused by enzymes (nucleases), to reduce uptake and degradation by immune cells (macrophages) and to prevent non-specific uptake by cells. We believe that introducing protein-binding domains in oligonucleotides can be used as a strategy to control the fate of oligonucleotides in physiological environments. While our study focuses on albumin, we believe that such systematic studies, which elucidate the role of serum proteins systematically, will ultimately provide a toolbox to engineer the next-generation of therapeutic oligonucleotides, overcoming many of the barriers encountered by these therapeutics, such as stability, immunogenicity and off-target effects.

Design and Application of a Short (16-mer) Locked Nucleic Acid Splice-Switching Oligonucleotide for Dystrophin Production in Duchenne Muscular Dystrophy Myotubes.

Splice-switching oligonucleotides (SSOs) have been used to modulate gene expression by interfering with pre-mRNA splicing with the intent to treat disease. For Duchenne muscular dystrophy, splicing modulation has been used to induce the skipping of exon 51 of the dystrophin transcript, allowing the production of a truncated but functional protein. Although oligonucleotide-based therapies are promising, the rapid degradation of oligonucleotides (ONs) by intracellular nucleases has been a major obstacle. Locked nucleic acid (LNA) substitution in SSOs protects oligonucleotides from nuclease degradation and enhances the hybridization properties of the oligo. However, the best optimum size of the oligo depends on the LNA substitution rate. Here we show that 16-mer DNA SSOs with 60% LNA substitution and full phosphorothioate (PS) linkage backbone efficiently induce exon 51 skipping in myogenic cells derived from a DMD patient, allowing expression of the dystrophin protein.

Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

Novel in vivo excision assays for monitoring the excised oligonucleotide products of nucleotide excision repair in UV-irradiated cells have provided unprecedented views of the kinetics and genomic distribution of repair events. However, an unresolved issue is the fate of the excised oligonucleotide products of repair and their mechanism of degradation. Based on our observation that decreases in excised oligonucleotide abundance coincide with the induction of apoptotic signaling in UV-irradiated cells, we considered the possibility that caspase-mediated apoptotic signaling contributes to excised oligonucleotide degradation or to a general inhibition of the excision repair system. However, genetic and pharmacological approaches to inhibit apoptotic signaling demonstrated that caspase-mediated apoptotic signaling does not affect excision repair or excised oligonucleotide stability. Nonetheless, our assay for detecting soluble DNAs produced by repair also revealed the production of larger DNAs following DNA damage induction that was dependent on caspase activation. We therefore further exploited the versatility of this assay by showing that soluble DNAs produced by both nucleotide excision repair and apoptotic signaling can be monitored simultaneously with a diverse set of DNA damaging agents. Thus, our in vivo excision repair assay provides a sensitive measure of both repair kinetics and apoptotic signaling in genotoxin-treated cells.

Heat flux across an open pore enables the continuous replication and selection of oligonucleotides towards increasing length.

The replication of nucleic acids is central to the origin of life. On the early Earth, suitable non-equilibrium boundary conditions would have been required to surmount the effects of thermodynamic equilibrium such as the dilution and degradation of oligonucleotides. One particularly intractable experimental finding is that short genetic polymers replicate faster and outcompete longer ones, which leads to ever shorter sequences and the loss of genetic information. Here we show that a heat flux across an open pore in submerged rock concentrates replicating oligonucleotides from a constant feeding flow and selects for longer strands. Our experiments utilize the interplay of molecular thermophoresis and laminar convection, the latter driving strand separation and exponential replication. Strands of 75 nucleotides survive whereas strands half as long die out, which inverts the above dilemma of the survival of the shortest. The combined feeding, thermal cycling and positive length selection opens the door for a stable molecular evolution in the long-term microhabitat of heated porous rock.

Design, synthesis and properties of artificial nucleic acids from (R)-4-amino-butane-1,3-diol

A new artificial nucleic acid analogue, (R)-Am-BuNA, was developed with a simplified acyclic (R)-4-amino-butane-1,3-diol phosphodiester backbone. Phosphoramidite monomers of (R)-Am-BuNA were incorporated into DNA oligonucleotides (ODNs) and G-quadruplexes. Their thermal stability, conformation change and biological stability were further investigated using UV-melting, circular dichroism (CD) and gel electrophoresis. The results suggested that thermal stability of the duplexes of (R)-Am-BuNA modified ODNs and their complementary ODN is highly dependent on the substitution position. Substitution of thymidine at the 7th position in a thrombin-binding DNA aptamer (TBA) results in a slight increase in Tm with no effect on quadruplex conformation on the CD spectrum in comparison to that of the natural G-quadruplex. Further enzymatic experiments with fetal bovine serum (FBS) and snake venom phosphodiesterase (SVPDE) indicated that only single replacement of a (R)-Am-BuNA modified nucleobase greatly inhibited oligonucleotide degradation, which shows their promising applications as capping nucleotides in nucleic acid drugs.

Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering

Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

Fluorescence correlation spectroscopy to study the degradation of oligonucleotides, both naked and complexed, to cationic carriers

The degradation of double-labeled oligonucleotides (ONs) was followed by the disappearance of Fluorescence Energy Transfer between the rhodamine green and Cy5 fluorophores attached to the 3′ and 5′ end of the oligonucleotides. The fluorescence intensities after rhodamine green excitation were recorded using the ultra-sensitive detectors of a dual-color FCS setup. The ratio of the Cy5 to rhodamine green fluorescence showed to give accurate information on the integrity of the oligonucleotides and could be easily used to follow the degradation of naked or complexed double-labeled oligonucleotides, both in buffer and in living cells.

Nucleotide Pyrophosphatase/Phosphodiesterase 1 Is Responsible for Degradation of Antisense Phosphorothioate Oligonucleotides

The rapid degradation of unmodified phosphodiester oligodeoxynucleotides (PO-oligos) by exo -and endonucleases limits their application as antisense constructs and requires the synthesis and use of modified oligonucleotides. Phosphorothioate analogs of oligonucleotides (PS-oligos) are much more stable against nucleolytic degradation than their unmodified counterparts, and this is one of the reasons for which they are a promising class of antisense oligonucleotides. However, PS-oligos also undergo slow hydrolysis by enzymes present in plasma. The oligonucleotide degradation proceeds mainly from the 3' -end, resulting in the formation of a typical ladder of shorter products and the release of the mononucleoside 5' -phosphorothioates. So far, little has been known concerning the molecular identity of the enzymes involved in the degradation of PS-oligos. We now identify the human plasma 3' -exonuclease responsible for their degradation as a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9), also known as the plasma cell differentiation antigen PC-1. We also show that adenosine or deoxyadenosine (alpha-thio)triphosphates can act as potent inhibitors of NPPs.

Molecular and Cellular Barriers Limiting the Effectiveness of Antisense Oligonucleotides

Antisense oligonucleotides present a powerful means to inhibit expression of specific genes, but their effectiveness is limited by factors including cellular delivery, biochemical attack, and poor binding to target. We have developed a systems model of the processes required for an antisense oligonucleotide to enter, gain access to its target mRNA, and exert activity in a cell. The model accurately mimics observed trends in antisense effectiveness with the stability of the oligonucleotide backbone and with the affinity/kinetics of binding to the mRNA over the time course of inhibition. By varying the model parameters within the physically realizable range, we note that the major molecular and cellular barriers to antisense effectiveness are intracellular trafficking, oligonucleotide-mRNA binding rate, and nuclease degradation of oligonucleotides, with a weaker dependence on total cellular uptake than might be expected. Furthermore, the model may serve as a predictive tool to design and test strategies for the cellular use of antisense oligonucleotides. The use of integrated mathematical modeling can play a significant role in the development of antisense and related technologies.

Involvement of active oxygen species in protein and oligonucleotide degradation induced by nitrofurans

It is of great interest to know how nitrofurans are mutagenic and clastogenic. In particular, the 3-amino-2-oxazolidone (AOZ) ring, deriving from a cleavage of furazolidone, is not further metabolized and has been found to be part of protein-bound residues in edible tissues of farm animals and these might be released in the stomach of the consumer. The data in this paper show that isoniazide as well as AOZ and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), the latter deriving from furaltadone, cause irreversible damage to the prosthetic group of enzymes as well as degrade their polypeptide chain and cause fragmentation of the backbone chain of cellular or isolated DNA and RNA. Cellular DNA was degraded into small fragments of 2000 Mb, while rRNA was completely destroyed. Nitrofuran derivatives and hydrazides, in fact, share an N-N moiety, which is assumed to play an essential role in the irreversible damage observed. The key to the molecular mechanisms by which these compounds cause their irreversible effects may lie in oxygen consumption and electron spin resonance measurements, which reveal that both nitrofurans and isoniazide produce oxygen radicals at various degrees of efficiency. AOZ and AMOZ are not metabolized into more reactive metabolites, being themselves able to react with atmospheric oxygen and induce protein and oligonucleotide damage. The reaction does not require metal ions, although their presence will accelerate it.

A Dynamical Systems Model to Simulate the Perturbation Kinetics of Gene Expression by Antisense Oligonucleotides

Antisense oligonucleotides owe their efficacy to an ability to induce RNase H-dependent suppression of RNA translation, for sufficient time to allow physiological proteolysis. The magnitude and time delay preceding the protein nadir concentration determine the extent and timing of maximum antisense oligonucleotide activity. Antisense oligonucleotide degradation underlies reversal of RNA downregulation. The kinetics of protein downregulation is therefore determined by the complex interaction of both ligand chemistry (nuclease stability, affinity and RNase H activation), and gene expression kinetics. Optimization of antisense oligonucleotide efficacy and experimental design requires understanding of these interactions. The kinetics of protein and RNA downregulation have therefore been simulated by analysing a two-compartment kinetic model incorporating RNase H-dependent transcript degradation. The system of nonlinear differential equations describing this model was solved numerically using Runge–Kutte integration. The timecourse solutions corresponding to the four state variables (RNA, protein, antisense/RNA heteroduplex and antisense oligonucleotide), were determined simultaneously. This allowed systematic in silico examination of the consequences of altering variables such as oligonucleotide concentration, affinity, and stability, or the scheduling of multiple transfections on RNA and protein perturbations. By providing a tool for examining antisense oligonucleotide action theoretically, this heuristic model should facilitate both the rational design and interpretation of antisense experiments.

Integration of phosphodiesterase-induced degradation of oligonucleotides with capillary polymer-sieving electrophoresis.

Synthetic oligodeoxyribonucleotides (ODN) have been proposed as a class of potential therapeutic agents that can interact in a rational way with DNA or RNA, with the aim of inhibiting the expression of unwanted genetic information (, , ). One of the most critical questions in the evaluation of these molecules, is their stability toward enzymatic breakdown by [3′ or 5′]-exonucleases and endonucleases (). In order to inhibit or at least limit the effect of these nucleases, chemically modified ODNs have been synthesized.

Selective degradation of targeted mRNAs using partially modified oligonucleotides

At a theoretical level, the utilization of an oligonucleotide complementary to a target message to prevent translation sounds extraordinarily simple. There are, however, significant hurdles to the use of antisense oligonucleotides in vivo. Issues of nuclease stability and RNase H activity, which are presented in this chapter, must be considered in any discussion involving antisense oligonucleotides. A third important issue, cellular uptake of oligonucleotides, has not been discussed. This chapter has circumvented this complex and poorly understood area by directly microinjecting chimeric oligonucleotides into single cells. The ability to microinject oligonucleotides into Xenopus oocytes and embryos has made this an ideal system to ask important mechanistic questions involving oligonucleotide metabolism and mode of action. It is not possible, for example, to determine the rate of intracellular oligonucleotide degradation in cell or tissue culture because the rate of internalization is generally unknown. Additionally, it is not possible to compare the efficacy of two oligonucleotides adequately if they have different rates of internalization.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides.

The goal of the present paper was to encapsulate oligonucleotides in a new particulate carrier in order to protect them from enzymatic degradation. Nanocapsules with an aqueous core containing oligonucleotides were prepared by interfacial polymerization of isobutylcyanoacrylate in a W/O emulsion. Ultracentrifugation and re-suspension in water yielded a dispersion of these containing an aqueous core nanocapsules. Zeta potential measurements and quenching of fluorescence of fluorescein-bounded oligonucleotides were used to study the localization of the oligonucleotides. Oligonucleotide degradation studies were carried out in fetal calf serum. Polydisperse nanocapsules of size ranging from 20 to 400 nm were obtained. Oligonucleotide loading did not significantly influence the zeta potential, suggesting they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the oligonucleotides were efficiently protected from degradation by nucleases, whereas oligonucleotides adsorbed onto nanospheres were protected less efficiently. This paper describes, for the first time, a nanotechnology able to encapsulate oligonucleotides rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

In-Line Coupling of the Enzymatic Degradation of Oligonucleotides with Capillary Polymer Sieving Electrophoresis

This paper deals with how an enzymatic reaction can be integrated in a capillary electrophoretic system when the capillary is filled with a polymer solution. The difference in electrophoretic mobility between enzyme and substrate makes two zones merge inside the capillary. The feasibility of this integrated system is demonstrated by performing and following the degradation of short model homo-oligomeric deoxynucleotides (dA21 and pdA21) with phosphodiesterase I. After 3‘-exonucleolytic breakdown, oligonucleotide fragments are separated in-line in a 4% solution of a low viscosity-grade hydroxyethyl cellulose. Degradation and separation are achieved under 25 min, the number of manipulation steps is strongly reduced, and the system is fully automated. This technique opens possibilities of screening larger libraries of synthetic oligonucleotides for individual stability toward purified nucleases.

Fluorescence resonance energy transfer analysis of the degradation of an oligonucleotide protected by a very stable hairpin.

In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.

On-capillary enzymatic degradation of oligonucleotides studied by capillary electrophoresis in a non-gel sieving matrix

On-capillary enzymatic degradation of oligonucleotides studied by capillary electrophoresis in a non-gel sieving matrix

Stability of antisense oligonucleotides during incubation with a mixture of isolated lysosomal enzymes

Cellular uptake of antisense oligonucleotides occurs predominantly via the endocytic route but the subsequent intracellular trafficking and metabolic fate is still poorly understood. We have examined the stability of 20-mer phosphodiester (D-oligo) and phosphorothioate (S-oligo) oligonucleotides ([ 32P]-radiolabelled either internally and at the 3′- or 5′- end) during exposure to a mixture of isolated rat liver lysosomal enzymes (Tritosomes). Whereas the oligonucleotides remained stable in aqueous buffer at pH 5.4 for at least 48 h, the D-oligos (at 10 μM) were degraded relatively rapidly ( t 50% = 30–40 min) by Tritosomes and at a rate similar to S-oligos ( t 50% = 50 min). Degradation of both was verified using reverse-phase HPLC and in this case for D- and S-oligos (2 μM) the t 50% was 90 min and 150 min, respectively. Enzymatic digestion by lysosomal nucleases, and not simple acid-catalysed hydrolysis, accounts for the rapid degradation of oligonucleotides.