Role of locked nucleic acid modified complementary strand in quadruplex/Watson-Crick duplex equilibrium

In the human genome, the G-rich sequences that form quadruplexes are present along with their C-rich complementary strands; this suggests the existence of equilibrium between a quadruplex and a Watson-Crick duplex which allows the execution of their respective biological functions. We have investigated the sensitivity of this equilibrium to pharmacological agents by employing locked nucleic acid (LNA) modified complementary strands, and demonstrated successful invasion of the stable telomeric quadruplex d[(G(3)TTA)(3)G(3)]. Fluorescence, UV, ITC, and SPR studies were performed to understand the binding process involving the preformed quadruplex and LNA-modified complementary strands compared with that involving the unmodified complementary strand. Our data indicate that LNA modifications in the complementary strand shift the equilibrium toward the duplex state. These modifications confer increased thermodynamic stability to the duplex and increase the magnitude of relative free energy (DeltaDeltaG degrees) difference between duplex and quadruplex, thus favoring the predominance of duplex population over quadruplex. This superior ability of LNA-modified complementary strand can be exploited to pave an exploratory approach in which it hybridizes to a telomeric quadruplex and drives duplex formation, and inhibits the recognition of 3' G-rich overhang by RNA template of telomerase which guides telomere extension.     

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.     

Electron transfer of plurimodified DNA SAMs

An STM-based current-voltage (I/V) investigation of deoxyribonucleic acid (DNA) 18 base pair (bp) oligonucleotide monolayers on gold is presented. Three bases of each of the immobilized and complementary strands were modified with either iodine or phenylethylene moieties. The oligonucleotides were immobilized on template stripped gold (tsg) surfaces and characterized by atomic force microscopy (AFM) and scanning tunneling microscopy (STM). AFM imaging showed that monolayers of the expected height were formed. A comparative study of normal, halogenated, and phenyl-modified DNA was made with the STM in tunneling spectroscopy (TS) mode. I/V spectroscopic measurements in the range +/-250 mV on both single- and double-stranded (ds) DNA monolayers (modified and unmodified) showed that for negative substrate bias (U(sub)) electron transfer is more efficient through a phenyl-modified monolayer than through normal or halogenated DNA. This effect was particularly clear below a threshold bias of -100 mV. For positive U(sub), unmodified ds DNA was found to conduct slightly better than the modified strands. This is presumably caused by greater order in the unmodified versus modified DNA monolayers. Modifications on the immobilized (thiolated) strand seem to improve electron transport through the DNA monolayer more than modifications on the complementary (not surface-bound) strand.     

Oligo(nucleoside Phosphorothioate)s: The Quest of P-Chirality

The influence of the sense of chirality at phosphorus of internucleotide phosphorothioate groups in oligo(nucleoside phosphorothioate)s is discussed in terms of stability of stereodefined PS-Oligos in the intra- and intercellular media and newly discovered exclusive ability of R P -PS-Oligos containing homopurine tracts towards the formation of triple-stranded species of one phosphorothioate and two complementary RNA strands stechiometry.     

Synthesis of 2'-O-[2-(N-methylcarbamoyl)ethyl]ribonucleosides using oxa-Michael reaction and chemical and biological properties of oligonucleotide derivatives incorporating these modified ribonucleosides

To develop oligonucleotides containing new 2'-O-modified ribonucleosides as nucleic acid drugs, we synthesized three types of ribonucleoside derivatives modified at the 2'-hydroxyl group with 2-(methoxycarbonyl)ethyl (MOCE), 2-(N-methylcarbamoyl)ethyl (MCE), and 2-(N,N-dimethylcarbamoyl)ethyl (DMCE) groups, as key intermediates, via the oxa-Michael reaction of the appropriately protected ribonucleoside (U, C, A, and G) derivatives. Among them, the 2'-O-MCE ribonucleosides were found to be the most stable under basic conditions. To study the effects of the 2'-O-modification on the nuclease resistance of oligonucleotides incorporating the 2'-O-modified ribonucleosides and their hybridization affinities for the complementary RNA and DNA strands, 2'-O-MCE-ribonucleoside phosphoramidite derivatives were successfully synthesized and subjected to the synthesis of 2'-O-MCE-oligonucleotides and 2'-O-methyl-oligonucleotides incorporating 2'-O-MCE-ribonucleosides. The 2'-O-MCE-oligonucleotides and chimeric oligomers with 2'-O-MCE and 2'-O-methyl groups thus obtained demonstrated complementary RNA strands and much higher nuclease resistances than the corresponding 2'-O-methylated species. Finally, we incorporated the 2'-O-MCE-ribonucleosides into antisense 2'-O-methyl-oligoribonucleotides to examine their exon-skipping activities in splicing reactions related to pre-mRNA of mouse dystrophin. The exon-skipping assay of these 2'-O-methyl-oligonucleotide incorporating 2'-O-MCE-uridines showed better efficacies than the corresponding 2'-O-methylated oligoribonucleotide phosphorothioate derivatives.     

Evaluation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.     

A new route to 2'-O-alkyl-2-thiouridine derivatives via 4-O-protection of the uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside containing a 5'-CH(2)-extended chain at the sugar moiety was synthesized, thus isoadenosine and isothymidine were incorporated into a DNA single strand and siRNA. It was found that isonucleoside modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside () at 3' or 5' terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.     

In vitro metabolism of 2′-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC–tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3′-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5′-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.     

Applications of synthetic oligoribonucleotide analogues in studies of RNA structure and function

The study of RNA structure and function has been considerably aided by the development of methods for the chemical synthesis of oligoribonucleotides into which have been incorporated modified nucleosides carrying site-specific alterations. Such modifications are designed to eliminate or alter individual functional groups in the RNA which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparison of the properties of the modified RNA with unmodified RNA models allows conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA. The methods have been applied to studies of RNA structure, RNA catalysis, and interactions of RNA with proteins.     

Chemical synthesis of stimuli-responsive guide RNA for conditional control of CRISPR-Cas9 gene editing

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for conditional control of CRISPR-Cas9 functions are under active research, such as stimuli-responsive guide RNA (gRNA). However, the types of physiologically relevant stimuli that can trigger gRNA are largely limited due to the lack of a versatile synthetic approach in chemistry to introduce diverse labile modifications into gRNA. In this work, we developed such a general method to prepare stimuli-responsive gRNA based on site-specific derivatization of 2′-O-methylribonucleotide phosphorothioate (PS-2′-OMe). We demonstrated CRISPR-Cas9-mediated gene editing in human cells triggered by oxidative stress and visible light, respectively. Our study tackles the synthetic challenge and paves the way for chemically modified RNA to play more active roles in gene therapy.

Conditional control of CRISPR-Cas9 activity by reactive oxygen species and visible light is achieved using stimuli-responsive guide RNA synthesized by a general method based on RNA 2′-O-methylribonucleotide phosphorothioate.     

In vitro Selection of Chemically Modified DNAzymes

DNAzymes are in vitro selected DNA oligonucleotides with catalytic activities. RNA cleavage is one of the most extensively studied DNAzyme reactions. To expand the chemical functionality of DNA, various chemical modifications have been made during and after selection. In this review, we summarize examples of RNA-cleaving DNAzymes and focus on those modifications introduced during in vitro selection. By incorporating various modified nucleotides via polymerase chain reaction (PCR) or primer extension, a few DNAzymes were obtained that can be specifically activated by metal ions such as Zn²⁺ and Hg²⁺. In addition, some modifications were introduced to mimic RNase A that can cleave RNA substrates in the absence of divalent metal ions. In addition, single modifications at the fixed regions of DNA libraries, especially at the cleavage junctions, have been tested, and examples of DNAzymes with phosphorothioate and histidine-glycine modified tertiary amine were successfully obtained specific for Cu²⁺, Cd²⁺, Zn²⁺, and Ni²⁺. Labeling fluorophore/quencher pair right next to the cleavage junction was also used to obtain signaling DNAzymes for detecting various metal ions and cells. Furthermore, we reviewed work on the cleavage of 2′-5′ linked RNA and L-RNA substrates. Finally, applications of these modified DNAzymes as biosensors, RNases, and biochemical probes are briefly described with a few future research opportunities outlined at the end.     

Spermine moiety attached to the C-5 position of deoxyuridine enhances the duplex stability of the phosphorothioate DNA/complementary DNA and shows the susceptibility of the substrate to RNase H

Novel phosphorothioate-modified oligodeoxynucleotides (S-ODNs) containing a deoxyuridine derivative bearing a spermine moiety at the C-5 position were synthesized. The study of the thermal stability and the thermodynamic stability showed that the modified S-ODNs have been able to form the stable duplexes with the complementary DNA. It was also found that the duplex composed of the modified S-ODN and its complementary RNA strand is the substrate for Escherichia coli RNase H, and the cleavage of the RNA strand by the enzyme was almost similar as in the case of the unmodified one.     

Binary malachite green aptamer for fluorescent detection of nucleic acids

A new probe that can fluorescently report the presence of specific nucleic acids in solution with extremely high selectivity was developed. The probe consists of malachite green-a triphenylmethane dye-and two short RNA strands, each of which comprises a fragment complementary to an analyte molecule and a fragment of a malachite green aptamer (MGA). The two RNA strands form MGA upon hybridization to the adjacent positions of the nucleic acid analyte. MGA is able to bind malachite green and enhance the fluorescence of the dye, thus monitoring the presence of the nucleic acid in solution. The probe reliably discriminates against 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at room temperature in physiological buffer. Consisting of unmodified RNA strands, which can be expressed in living cells, binary MGA probe represents a promising instrument for real-time nucleic acid monitoring in vivo.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.     

Synthesis and hybridization properties of l-oligodeoxynucleotide analogues fixed in a low anti glycosyl conformation

We have synthesized l-type enantiomers (cU and cA) of nucleoside analogues, whose glycosyl bonds are fixed in a low anti conformation (ap glycosyl conformation, [small chi][approximate] 180[degree]), and incorporated them into oligonucleotides to evaluate the hybridization ability with natural DNA and RNA sequences. Although the incorporation of the modified nucleosides into oligonucleotides decreased the hybridization ability with unmodified complementary DNA sequences, the fully-substituted 12mers (cU(12) and cA(12)) still retained the hybridization ability with the complementary unmodified DNA 12mers, regardless of their unnatural l-chirality. In contrast, cU(12) and cA(12) showed different hybridization behavior with complementary unmodified RNA 12mers. cU(12) forms a more stable duplex with rA(12) than the corresponding natural 12mer (dT(12)), whereas cA(12) cannot hybridize with rU(12). Based on the model structure of cU(12)-rA(12), we discuss these experimental results.     

Chimeric oligonucleotides based on 2"-O-modified oligoribonucleotides with the terminal 3"—3" internucleotide linkage as potential inhibitors of MDR 1 gene expression

Chimeric constructs were synthesized based on oligoribonucleotides modified at the 2"-position of the ribose (2"-O-tetrahydropyranyl- or 2"-O-methyl-) and at the 3"-terminus of the oligonucleotide chain (terminal 3"—3" internucleotide linkage), which are complementary to a region of MDR 1 mRNA. A comparative study of the properties of these chimeric constructs was performed. The chimeric oligomers with the modified 3"-terminus are characterized by high stability with respect to 3"-exonucleases, form stable complementary complexes with RNA, and can activate RNase H in a duplex with RNA.     

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.