Novel photocleavable universal support for oligonucleotide synthesis

A novel photocleavable universal support for the automated solid phase synthesis of oligonucleotides is described. The linker between the growing oligonucleotide chain and CPG support contains a nucleophilic amine protected with a photocleavable group. On exposure to UV light, this group is detached and the free amine affords cleavage of the oligonucleotide from the support. The use of long wavelength UV light avoids damage to the DNA.     

Synthesis of High Quality Phosphorothioate Oligonucleotides as Antisense Drugs. Use of I-Linker in the Elimination of 3′-Terminal Phosphorothioate Monoesters

Abstract

Detritylation of a 5′-O-DMT-2′-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3′-terminal phosphorothiaote (3′-TPT) monoester (n?1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3′-TPT formation thereby improving the quality of oligonucleotide synthesized.     

Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.

We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.     

Introduction of 5′-Terminal Amino and Thiol Groups into Synthetic Oligonucleotides

Abstract

Oligonucleotides terminating in a 5′-primary amine group are synthesized using solid phase phosphoramidite chemistry. The 5′-terminal amine group in the deprotected oligonucleotide is further derivatized with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) followed by treatment with dithiothreitol (DTT) to produce 5′-thiol terminated oligonucleotides. Introduction of 5′-thiol group is further confirmed by reading the absorbance of the released chromophore, pyridine-2-thione at 343 nm; ?₃₄₃=8080/M.     

Facile and Rapid Deprotection Conditions for the Cleavage of Synthetic Oligonucleotides from 1,4-Anhydroerythritol-Based Universal Polymer Support

In our previous report [Kumar, P.; Dhawan, G.; Chandra, R.; Gupta, K.C. Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support. Nucl. Acids Res. 2002, 30, e130 (1-8)], we demonstrated polyamine-mediated deprotection of oligonucleotides from cis-diol group bearing universal polymer support (I). However, vulnerability of the conventional dC^bz to modifications under these conditions compelled us to employ dC^ac during synthesis of oligonucleotide using conventional synthons. Here, a new set of simple and rapid deprotection conditions has been developed for the complete cleavage of oligonucleotides from the 1,4-anhydroerythritol-based universal polymer support employing conventional dC^bz synthon. Using manganese-imidazole complex in aqueous ammonium hydroxide (～30%), fully deprotected oligonucleotide sequences were obtained in 40 min, which were analyzed on reverse phase-HPLC and compared with the standard oligomers in terms of their retention time. Finally, their biological compatibility was established by analyzing PCR amplified products of npsA gene of N. meningitidis.     

Synthesis and Binding Properties of Oligonucleotides Containing an Azobenzene Linker

Abstract

Incorporation of an azobenzene-4,4′-diamide group via a linker arm into the 3′-hydroxyl function of one oligonucleotide segment and the 5′-OH of other oligonucleotide has been described. The binding of the oligonucleotides containing the azobenzene linker was investigated by UV melting behaviors. The azobenzene linker has been shown to be useful as an effective bridge for stabilizing hairpin duplex and triplex.     

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.     

早期肠内营养支持在根治性膀胱全切患者术后的应用研究

目的:探讨根治性膀胱全切术后的最佳营养支持方法。方法:采用随机数法将60例根治性膀胱全切患者随机分为肠外营养支持(PNS)组和肠内营养支持组(ENS),比较两组患者的营养时间、术后下床时间以及住院时间、营养支持费用、血清总蛋白、白蛋白、前白蛋白以及术后并发症的发生情况。结果:术后7 d,ENS组血清总蛋白、白蛋白及前白蛋白均显著高于PNS组(p0.05)。ENS组的营养时间、术后下床时间以及住院时间均短于PNS组(P0.05),且营养支持费用少于PNS组(P0.05)。两组患者术后均未发生肠梗阻等手术相关并发症,PNS组发生静脉炎3例。结论:早期肠内营养是根治性膀胱全切患者术后理想的营养支持模式。     

The p-Phenylazophenyloxycarbonyl Protecting Group: Selective Deblocking and Oligonucleotide Synthesis Avoiding Acid Steps

Abstract

The p-phenylazophenyloxycarbonyl group is selectively introduced at nucleoside-5′-OH by reaction with the resp. chloroformate and deblocked e.g. by transesterification/β-elimination. With it we designed a reaction cycle for oligonucleotide support synthesis, that avoids acid deprotection.     

Molecular Modeling Study on the Enantioselective Binding of S- and R-Ofloxacin to Various DNA Sequences

Abstract

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT]₂, d[GCGATATCGC]₂ and d[ATAICICTAT]₂, were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT]₂ complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.     

Photoregulation of DNA polymerase I (Klenow) with caged fluorescent oligodeoxynucleotides

The DNA polymerase reaction by Klenow fragment (KF) was efficiently regulated with UV light using a 25-mer caged fluorescent oligodeoxynucleotide (CFO) as the template. The CFO was functionalized with a fluorescein reporter (Fl) and photocleavable DABSYL quencher moiety (Dab). With Fl and Dab at adjacent cytidines in the middle at the template, KF was blocked from extending a complementary 12-mer primer. Upon UV photolysis of the DABSYL blocking group under aerobic conditions, fluorescein emission was restored and 50% of the primers were fully extended by KF.     

Preactivated carboxyl linker for the rapid conjugation of alkylamines to oligonucleotides on solid support

A C10 linker phosphoramidite reagent terminated with a succinimidyl-activated carboxyl group was prepared and used to couple to the 5'-end of an oligonucleotide synthesized on a solid support. The succinimidyl-activated carboxyl functionality can be used for rapid conjugation of amines to oligonucleotides on solid support or it can be hydrolyzed to form a carboxylic acid functionality. The activated linker was successfully used for conjugation of several primary and secondary aliphatic amine derivatives (including biotin and fluorescein cadaverine) onto a solid support-bound 12-mer DNA oligonucleotide at scales ranging from 0.15 to 1.0 micromol. The overall yields of the conjugation products after AMA deprotection and cleavage from the solid support ranged from 43 to 75% of the total oligonucleotide product. This value is significant, as it includes oligonucleotide synthesis, coupling of the linker, and conjugation of the amine. In addition, the entire process of oligonucleotide synthesis, linker coupling, amine conjugation, deprotection, and cleavage of the oligonucleotide from solid support can be accomplished in 1 day.     

Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)

Abstract

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs’ analogs have synthesized by structural changes of nucleoside’s susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl – analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π–π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.     

Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.

The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.     

Express Protocol for Functionalization of Polymer Supports for Oligonucleotide Synthesis

Abstract

A rapid and general one-pot method is described for the attachment of the leader nucleoside onto the polymer supports, suitable for polymer supported oligonucleotide synthesis following oxidation-reduction condensation. The method can also be used for support functionalisation in fully automated DNA synthesizer prior to oligonucleotide synthesis.     

Postsynthetic Transformations of Oligodeoxynucleotides Originated at 6-Methylthio-purine Site

Abstract

New route to oligodeoxynucleotides labeled with fluorescent luminarine was explored. Regioselective oxidation of 6-methylthio-purines to 6-methylsulphoxides reactive toward pyridine was achieved. Upon UV irradiation of 6-pyridinium-purines oligonucleotide cleavage instead of phototransformation to luminarine was observed.     

Stabilization and photochemical regulation of antisense agents through PEGylation

Oligonucleotides are effective tools for the regulation of gene expression in cell culture and model organisms, most importantly through antisense mechanisms. Due to the inherent instability of DNA antisense agents, various modifications have been introduced to increase the efficacy of oligonucleotides, including phosphorothioate DNA, locked nucleic acids, peptide nucleic acids, and others. Here, we present antisense agent stabilization through conjugation of a poly(ethylene glycol) (PEG) group to a DNA oligonucleotide. By employing a photocleavable linker between the PEG group and the antisense agent, we were able to achieve light-induced deactivation of antisense activity. The bioconjugated PEG group provides stability to the DNA antisense agent without affecting its native function of silencing gene expression via RNase H-catalyzed mRNA degradation. Once irradiated with UV light of 365 nm, the PEG group is cleaved from the antisense agent leaving the DNA unprotected and open for degradation by endogenous nucleases, thereby restoring gene expression. By using a photocleavable PEG group (PhotoPEG), antisense activity can be regulated with high spatial and temporal resolution, paving the way for precise regulation of gene expression in biological systems.     

Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the ‘next-generation’ technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N⁶-position of 2′-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3′-OH group of the N⁶-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N⁶-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.     

营养支持用于急性发作期慢性阻塞性肺疾病的效果研究

目的:探讨营养支持用于急性发作期慢性阻塞性肺疾病患者的临床效果。方法:选取2015年1月至2017年1月经新疆维吾尔自治区人民医院诊断的160例急性发作期慢性阻塞性肺疾病患者,随机分为两组,每组各80例。对照组仅给予抗炎、解痉、平喘等治疗;观察组在此基础上加以营养支持治疗。检测和比较两组治疗前后的肺功能指标、营养状况、血气指标、免疫指标、住院时间。结果:治疗后,观察组的肺功能指标第一秒用力呼气量(FEV)、第一秒用力呼气量与用力肺活量的百分比(FEV1%)、营养状况指标(血清白蛋白(ALB)、前白蛋白(PALB)、免疫功能指标(CD3~+、CD4~+、CD4~+/CD8~+)水平及外周淋巴细胞计数均较照组显著升高(P0.05)。此外,观察组的PO_2高于对照组,PCO_2较对照组降低,且住院时间均明显短于对照组(P0.05)。结论:及时进行营养支持治疗有助于改善急性发作期慢性阻塞性肺疾病患者的肺功能及免疫功能,并缩短住院时间。