Oligonucleotide chemistry has been developed greatly over the past three decades, with many advances in increasing nuclease resistance, enhancing duplex stability and assisting with cellular uptake. Locked nucleic acid (LNA) is a structurally rigid modification that increases the binding affinity of a modified-oligonucleotide. In contrast, unlocked nucleic acid (UNA) is a highly flexible modification, which can be used to modulate duplex characteristics. In this tutorial review, we will compare the synthetic routes to both of these modifications, contrast the structural features, examine the hybridization properties of LNA and UNA modified duplexes, and discuss how they have been applied within biotechnology and drug research. LNA has found widespread use in antisense oligonucleotide technology, where it can stabilize interactions with target RNA and protect from cellular nucleases. The newly emerging field of siRNAs has made use of LNA and, recently, also UNA. These modifications are able to increase double-stranded RNA stability in serum and decrease off-target effects seen with conventional siRNAs. LNA and UNA are also emerging as versatile modifications for aptamers. Their application to known aptamer structures has opened up the possibility of future selection of LNA-modified aptamers. Each of these oligonucleotide technologies has the potential to become a new type of therapy to treat a wide variety of diseases, and LNA and UNA will no doubt play a part in future developments of therapeutic and diagnostic oligonucleotides.
Background A decline in hospitalization for cardiovascular events and catheter laboratory activation was reported for the United States and Italy during the initial stage of the Covid-19 pandemic of 2020. We report on the deployment of emergency services for cardiovascular events in a defined region in western Germany during the government-imposed lock-down period. Methods We examined 5799 consecutive patients who were treated by emergency services for cardiovascular events during the Covid-19 pandemic (January 1 to April 30, 2020), and compared those to the corresponding time frame in 2019. Examining the emergency physicians’ records provided by nine locations in the area, we found a 20% overall decline in cardiovascular admissions. Results The greatest reduction could be seen immediately following the government-imposed social restrictions. This reduction was mainly driven by a reduction in discretionary admissions for dizziness/syncope (-53%), heart failure (-38%), exacerbated COPD (-28%) and unstable angina (-23%), while unavoidable admissions for ST-elevation myocardial infarction (STEMI), cardiopulmonary resuscitation (CPR) and stroke were unchanged. There was a greater decline in emergency admissions for patients ≥60 years. There was also a greater reduction in emergency admissions for those living in urban areas compared to suburban areas. Conclusions During the Covid-19 pandemic, a significant decline in hospitalization for cardiovascular events was observed during the government-enforced shutdown in a predefined area in western Germany. This reduction in admissions was mainly driven by “discretionary” cardiovascular events (unstable angina, heart failure, exacerbated COPD and dizziness/syncope), but events in which admission was unavoidable (CPR, STEMI and stroke) did not change.
Novel pyrene-perylene α-L-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally positioned FRET pairs is achieved with low to no fluorescence background signal, remarkably low limit of target detection values and stabilization of the resulting nanostructures.
Herein we introduce a novel fluorescent LNA/DNA machine, a nanocrawler, which reversibly moves along a directionally polar complementary road controlled by affinity-enhancing locked nucleic acid (LNA) monomers and additional regulatory strands. Polyaromatic hydrocarbon (PAH) dyes attached to 2'-amino-LNA monomers are incorporated at four stations of the system, enabling simple detection of the position of the nanocrawler via a step-specific color signal. The sensing is provided by highly sensitive, chemically stable, and photostable PAH LNA interstrand communication systems, including pyrene excimer formation and pyrene-perylene interstrand Förster resonance energy transfer. We furthermore demonstrate that the nanocrawler selectively and reversibly moves along the road, followed by a bright and consistent fluorescence response for up to 10 cycles without any loss of signal.
The cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) or cisplatin reacts primarily with guanines in DNA to form 1,2-Pt-GG and 1,3-Pt-GNG intrastrand cross-links, and to a lesser extent, G-G interstrand cross-links. Recent NMR evidence has suggested that cisplatin can also form a coordination complex with the phosphodiester internucleotide linkage of DNA. We have examined the effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a -GTG-sequence. Cisplatin forms a stable adduct with TpT that can be isolated by reversed phase HPLC. The cis-Pt-TpT adduct contains a single Pt, as determined by atomic absorption spectroscopy (AAS) and by electrospray ionization mass spectrometry (ESI-MS), and is resistant to digestion by snake venom phosphodiesterase. Treatment of the adduct with sodium cyanide regenerates TpT. Similar adduct formation was observed when T(pT) 8 was treated with cisplatin, but not when the phosphodiester linkages of T(pT) 8 were replaced with methylphosphonate groups. These results suggest that the platinum may be coordinated with the oxygens of the thymine and possibly with those of the phosphodiester group. As expected reaction of a 9-mer containing a -GTG-sequence with cisplatin yielded an adduct that contained a 1,3-Pt-GTG intrastrand cross-link. However, we found that the number and placement of phosphodiesters surrounding a -GTG-sequence significantly affected intrastrand cross-link formation. Increasing the number of negatively charged phosphodiesters in the oligonucleotide, increased the amount of -GTGplatination. Surrounding the -GTG-sequence with non-ionic methylphosphonate linkages reduced or eliminated cross-link formation. These observations suggest that interactions between cisplatin and the negatively charged phosphodiester backbone may play an important role in facilitating platination of guanine nucleotides in DNA.Reactions of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) with DNA have been studied for many years. Although it is widely accepted that the therapeutically relevant lesion is a 1,2-Pt-GG intrastrand cross-link, which links the N7s of the two guanines (1-3), the precise pathway leading to formation of this cross-link, as well as to 1,3-Pt-GNG intrastrand and G-G interstrand cross-links, is still under investigation. It is thought that cis-DDP is converted to cis-diamminediaquaplatinum(II) upon leaving the high concentration of chloride in the blood and entering the low concentration of chloride in the cell (3)(4)(5). This dicationic, aquated species then reacts with the DNA bases and other nucleophiles within the cell. While the reactions of cisplatin with nucleobases have been studied extensively, interactions with the DNA backbone have been largely overlooked. Platinum, a "soft" metal, preferentially binds to other "soft" ligands, such as amines and thiols (6,7). This binding preference does not, however, exclude the possibility that platinum can for...
Triplex-forming oligonucleotides (TFOs) can bind to polypurine•polypyrimidine tracts in DNA and as a consequence, perturb normal functioning of a targeted gene. The effectiveness of such anti-gene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here we report that attachment of N-7-platinated guanine nucleosides to the 3′-and/or 5′-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3′,5′ bis platinated TFOs can result in formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37°C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast high levels (~86%) of adduct formation by platinated 2′-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2′-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinumconjugated TFOs may be good candidates for use as anti-gene agents.Over the past two decades, there has been considerable interest in developing oligonucleotides that can specifically inhibit gene expression. Two approaches that have received much attention are antisense oligonucleotides and siRNA. Both approaches target the RNA product of the expressed gene and ultimately result in destruction of this target. Because the RNA products are continuously produced when the gene is transcribed, the antisense oligonucleotide or siRNA must be present constantly in order to prevent their translation into protein.An alternative approach that directly targets and inhibits gene expression utilizes triplexforming oligonucleotides (TFOs). These oligonucleotides are designed to bind to doublestranded DNA at specific regions within the gene (1-3 ). As a consequence of this binding, transcription of the gene is inhibited or mutations are induced that result in aberrant expression Triplex forming oligonucleotides interact via the major groove by binding to polypurine tracts within the DNA target (1-7 ). Oligopurine TFOs utilize reverse-Hoogsteen hydrogen bonds to form A•A-G and G•G-C triads and are oriented antiparallel to the polypurine tract. Oligopyrimidine TFOs utilize Hoogsteen hydrogen bonds to form T•A-T and C + •G-C triads and are oriented parallel to the polypurine tract. In this case the N-3 of the cytosine of the C + •G-C triad must be protonated in order to form a stable Hoogsteen interaction and consequ...
Enzymatic recognition of unlocked nucleic acid (UNA) nucleotides was successfully accomplished. Therminator DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of a 81 mer UNA-modified DNA library was efficiently achieved by KOD DNA polymerase.Development of nucleic acid therapeutics has attracted significant interest for the treatment of many diseases. With one aptamerbased drug, Macugen (Pegaptanib sodium), 1 on the market for the treatment of age-related macular degeneration (AMD), this class of nucleic acid constructs are an emerging attractive class of therapeutic molecules.2 Highly specific DNA or RNA aptamers with high binding affinity to their targets are normally selected from a large pool of oligonucleotides by in vitro selection processes.3 Chemically modified aptamers are used to improve pharmacodynamic, as well as pharmacokinetic properties. The application of modified nucleotides in the aptamer selection processes is rather limited due to their poor substrate specificities to polymerases. However, there are a few reports that describe the selection of aptamers in a single step with only one enzymatic protocol that involves polymerase chain reaction (PCR) amplification. 4 Establishing an efficient PCR method for a library containing modified nucleotides is a key step prior to successful selection of chemically modified aptamers.Unlocked nucleic acid (UNA) is an RNA mimic in which the bond between the C2 0 and C3 0 atoms of the ribose ring is cleaved, which results in high flexibility relative to the parent RNA monomer (Scheme 1).5 Thermodynamic stability of i-motif structures can be modulated by introducing UNA nucleotide monomers.6 Furthermore, UNA was found to be a useful modification in siRNA-based gene-silencing technology as it can protect the siRNAs from serum degradation and offer reduced off-target effects while retaining gene silencing potency. 7Most importantly, the applicability of UNA nucleotides in aptamer development was recently studied by systematically incorporating them in a known DNA aptamer against thrombin, whereby it was demonstrated that introduction of UNA monomers at specific positions significantly increased the binding affinity. 8 These remarkable findings clearly substantiate the need to develop UNA-modified aptamers from a large library by in vitro selection processes. Herein, for the first time, we report enzymatic recognition studies involving UNA nucleotides.We investigated UNA nucleotides as substrates for DNA polymerases in line with our current focus on one-step selection of UNA-modified DNA aptamers. First, we synthesized UNA-C nucleoside 5 0 -triphosphates in small scale according to a procedure published for nucleoside triphosphate synthesis, 9 after which all four UNA triphosphates (Fig. 1) were prepared in larger scale. Primer extension assays were carried out to screen six different DNA polymerases from both A-and B-families, including Therminator, KOD, Phusion, Pfu, Klenow, and Taq...
Polypyrimidine oligonucleotides can bind to tracts of contiguous purines in double-stranded DNA to form triple-stranded complexes. The stability of the triplex is reduced significantly if the target purine tract is interrupted by a single pyrimidine. Previous studies have shown that incorporation of an N4-aminoalkylcytosine into the triplex-forming oligonucleotide (TFO), opposite a single CG interruption, facilitates triplex formation. Examination of molecular models suggested that further modification of the amino group of the aminoalkyl arm might enable adduct formation with the N7 of the guanine of the CG interruption. To test this, we prepared 2′-deoxyribo-and 2′-O-methylribo-TFOs that contained cytosine (C), N4-(2-aminoethyl)cytosine (ae-C), or diethylenetriamineplatinum(II) (DPt-C) or cis-aquodiammineplatinum(II) (cPt-C) derivatives of N4-(2-aminoethyl)cytosine, positioned opposite a CG interruption of a polypurine tract found in the pol gene of HIV-1 proviral DNA. Although the C- and ae-C-derivatized deoxyribo-TFOs formed triplexes of modest stability and the DPt-C-modified TFO failed to form a triplex, the C- and ae-C-derivatized 2′-O-methylribo-TFOs formed remarkably stable triplexes (Tm = 57 °C). The DPt-C- and cPt-C-modified 2′-O-methylribo-TFOs also formed triplexes, although their stabilities were reduced (Tm = 33 °C), suggesting that the tethered platinum group may interfere sterically with TFO binding. Consistent with this hypothesis was the observation that triplex stability was restored (Tm = 57 °C) when the diethylenetriamineplatinum(II) group was tethered to the 5′-end of the 2′-O-methylribo-TFO via a 2-aminoethylcarbamate linkage. Taken together, these results suggest that 2′-O-methylribo-TFOs may be particularly useful in targeting purine tracts in DNA that have CG interruptions, and that further modification with platinum derivatives could lead to the design of TFOs that are capable of covalent binding to their target, thus increasing the effectiveness of the TFO.Key words: triplex-forming oligonucleotide, TFO, cisplatin, interrupted polypurine tract.
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