Long oligodeoxynucleotides: chemical synthesis, isolation via catching-by-polymerization, verification via sequencing, and gene expression demonstration

• Yipeng Yin,
• Reed Arneson,
• Alexander Apostle,
• Adikari M. D. N. Eriyagama,
• Komal Chillar,
• Emma Burke,
• Martina Jahfetson,
• Yinan Yuan and
• Shiyue Fang

Beilstein J. Org. Chem. 2023, 19, 1957–1965, doi:10.3762/bjoc.19.146

• . Keywords: automated synthesis; catching-by-polymerization; gene assembly; long oligonucleotide; synthetic biology; Introduction Long oligodeoxynucleotides (ODNs) are segments of DNAs extending beyond one hundred nucleotides (nt). Emerging research areas such as synthetic biology [1][2], protein

The role of chemistry in the success of oligonucleotides as therapeutics

• Pawan Kumar and
• Tom Brown

Beilstein J. Org. Chem. 2022, 18, 197–199, doi:10.3762/bjoc.18.22

• double-stranded nucleotides, which are called short interfering RNA or “siRNA,” can target mRNA and prevent it from being translated to make proteins [2]. While the mechanism by which antisense oligonucleotides (single stranded oligonucleotide) and siRNA (short RNA duplexes) work are completely different

• , both of them target mRNA to disrupt protein synthesis. In the following text we will refer both antisense oligonucleotides and siRNAs collectively as therapeutic oligonucleotides. More than 10 oligonucleotide drugs have received regulatory approval by the FDA and are now helping patients suffering from

• forefront of solving these issues, and have introduced many chemically modified nucleotides into oligonucleotides to increase their binding affinity toward RNA targets, and to improve their stability against nucleases to slow down degradation. This strategy has been successful, and most oligonucleotide

Cationic oligonucleotide derivatives and conjugates: A favorable approach for enhanced DNA and RNA targeting oligonucleotides

• Mathias B. Danielsen and
• Jesper Wengel

Beilstein J. Org. Chem. 2021, 17, 1828–1848, doi:10.3762/bjoc.17.125

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

• Nikita Brodyagin,
• Martins Katkevics,
• Venubabu Kotikam,
• Christopher A. Ryan and
• Eriks Rozners

Beilstein J. Org. Chem. 2021, 17, 1641–1688, doi:10.3762/bjoc.17.116

• suggesting that Io4 may be a promising alternative to T where stronger binding is desired [108]. PNA nucleobases for Hoogsteen recognition of pyrimidines: Triple helix formation, especially using tailored oligonucleotide analogues as PNA, could be a general and sequence specific approach for molecular

Double-headed nucleosides: Synthesis and applications

• Vineet Verma,
• Jyotirmoy Maity,
• Vipin K. Maikhuri,
• Ritika Sharma,
• Himal K. Ganguly and
• Ashok K. Prasad

Beilstein J. Org. Chem. 2021, 17, 1392–1439, doi:10.3762/bjoc.17.98

• followed by their duplex formation studies against complimentary oligonucleotide strands had described a very selective zipper-interaction [35], whereas a relative stabilization was observed due to stacking of these additional nucleobases across the minor groove [31]. The biological activity of the acyclic

• way junctions. There was a four-fold increase in the intensity of the pyrene excimer signal observed when an oligonucleotide containing two incorporations of the double-headed nucleoside 41 hybridized with an RNA target whereas the pyrene–pyrene excimer band almost vanished when the oligonucleotide

• then incorporated into oligonucleotide sequences, followed by thermal hybridization studies that indicated that the 5′-(S)-C-position is ideal for placing an additional nucleobase in the minor groove and interstrand stacking effects decreased with an increase in the length of the linker [31][65

Synthesis of 10-O-aryl-substituted berberine derivatives by Chan–Evans–Lam coupling and investigation of their DNA-binding properties

• Peter Jonas Wickhorst,
• Mathilda Blachnik,
• Denisa Lagumdzija and
• Heiko Ihmels

Beilstein J. Org. Chem. 2021, 17, 991–1000, doi:10.3762/bjoc.17.81

• as CD and LD spectroscopy. Fluorimetric DNA melting analysis with different types of quadruplex DNA revealed a moderate stabilization of the telomeric quadruplex-forming oligonucleotide sequence G3(TTAG3)3. The derivatives showed a moderate affinity towards quadruplex DNA (Kb = 5–9 × 105 M−1) and ct

• monitoring of the temperature-dependent Förster resonance energy transfer (FRET) between the dyes [46]. The particular oligonucleotide sequences were chosen because they are known to be involved in biologically relevant processes, namely in the transcription regulation of myc (FmycT) [47][48], kit (FkitT

• the most pronounced effect of the ligands on the ∆Tm values of F21T, the binding interactions with the corresponding unlabeled telomeric oligonucleotide sequence d[A(G3TTA)3G3] (22AG) were studied in more detail. Spectrometric titrations The interactions of derivatives 5a–e with calf thymus (ct) DNA

Beyond ribose and phosphate: Selected nucleic acid modifications for structure–function investigations and therapeutic applications

• Christopher Liczner,
• Kieran Duke,
• Gabrielle Juneau,
• Martin Egli and
• Christopher J. Wilds

Beilstein J. Org. Chem. 2021, 17, 908–931, doi:10.3762/bjoc.17.76

• Medicine, Vanderbilt University, Nashville, Tennessee 37232, United States 10.3762/bjoc.17.76 Abstract Over the past 25 years, the acceleration of achievements in the development of oligonucleotide-based therapeutics has resulted in numerous new drugs making it to the market for the treatment of various

• improving metabolic stability, pairing properties (RNA affinity), protein binding and transport/cellular uptake are concerned, chemical modifications are a prerequisite for the discovery and development of oligonucleotide therapeutics [11][12][13][14][15]. Thus, the natural PS and 2'-OMe backbone

• modifications provide improved resistance to degradation by exo- and endonucleases and they both affect protein binding [16][17]. Eight of the now approved 13 oligonucleotide drugs feature the PS modification in the backbone and all four approved siRNA therapeutics: ONPATTRO® (patisiran, 2018), GIVLAARI

Enhanced target cell specificity and uptake of lipid nanoparticles using RNA aptamers and peptides

• Roslyn M. Ray,
• Anders Højgaard Hansen,
• Maria Taskova,
• Bernhard Jandl,
• Jonas Hansen,
• Citra Soemardy,
• Kevin V. Morris and
• Kira Astakhova

Beilstein J. Org. Chem. 2021, 17, 891–907, doi:10.3762/bjoc.17.75

• exponential enrichment (SELEX) [30]. SELEX is an iterative process that begins with a large oligonucleotide library that, through a process of negative and positive selections, ends with a few candidates that are specific for a particular protein [30][31]. Using HIV-1 as our model, we explored the use of two

• (phosphate-buffered saline, PBS) conditions (Figure 4A). Additionally, we confirmed LNP uptake by the monocyte-derived macrophages (MDMs) using fluorescent microscopy (Figure 4B). We found that all LNPs containing the Cy5 oligonucleotide were observable under the microscope (Figure 4B) and that all

• , effective transport systems for brain drug delivery are highly warranted. Herein, we find that the LNP platform can be applied as a vehicle to circumvent the BBB and effectively deliver oligonucleotide probes to antigen-expressing cell lines. For HIV-1 there is currently a need for more effective delivery

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

• Yongdong Su,
• Maitsetseg Bayarjargal,
• Tracy K. Hale and
• Vyacheslav V. Filichev

Beilstein J. Org. Chem. 2021, 17, 749–761, doi:10.3762/bjoc.17.65

• the factors that determines the thermodynamic stability of nucleic acid secondary structures. Neutral or positively charged oligonucleotide analogues should bind more tightly with complementary DNA or RNA. Several studies have focused on the introduction of positively charged groups to a nucleobase

Synthesis and properties of oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing adenine, guanine, or 5-methylcytosine nucleobases

• Naohiro Horie,
• Takao Yamaguchi,
• Shinji Kumagai and
• Satoshi Obika

Beilstein J. Org. Chem. 2021, 17, 622–629, doi:10.3762/bjoc.17.54

• data indicate that GuNA[Me] could be a useful modification for therapeutic antisense oligonucleotides. Keywords: artificial nucleic acid; duplex-forming ability; oligonucleotide synthesis; Introduction The efficacy and safety of therapeutic oligonucleotides can be controlled by chemical modifications

• the middle position of 12-mer oligonucleotides (Table 1). The oligonucleotide synthesis was performed using an automated DNA synthesizer following the established synthetic method for GuNA[Me]-T-modified oligonucleotides [20]. 5-(Ethylthio)-1H-tetrazole (ETT) was used as an activator for the coupling

• ) at 60 °C for 5 h. Under these conditions, we obtained the GuNA[Me]-mC-modified oligonucleotide ON3 with high purity. In the case of the GuNA[Me] having a purine nucleobase (ON1 and ON2), the acetyl group in the guanidine moiety remained in a considerable amount. This means that we should give

Incorporation of a metal-mediated base pair into an ATP aptamer – using silver(I) ions to modulate aptamer function

• Marius H. Heddinga and
• Jens Müller

Beilstein J. Org. Chem. 2020, 16, 2870–2879, doi:10.3762/bjoc.16.236

• quadruplex, so that metal-mediated base pairs can be introduced. 3) It is a short oligonucleotide, so the modified aptamers can easily be synthesized using automated solid-phase synthesis. 4) The target molecule of this aptamer is a small molecule, facilitating the binding assays. 5) The aptamer also binds

• have Im:Im pairs inserted at different positions into the sequence (Figure 2). The oligonucleotide 1af is essentially identical to the established ATP/AMP-binding aptamer, with the attached fluorescein moiety representing the sole difference. The oligonucleotide 1bf bears one Im:Im pair located two

• to manipulate the formation of the aptamer:AMP complex. System A utilizes the 5’-fluorescein-labeled imidazole-containing oligonucleotide derived from the aptamer (1af, 1bf, 1cf, 1df) in combination with a complementary oligonucleotide (1q, quencher-labeled DNA) bearing a DABCYL moiety at the 3

Changed reactivity of secondary hydroxy groups in C8-modified adenosine – lessons learned from silylation

• Jennifer Frommer and
• Sabine Müller

Beilstein J. Org. Chem. 2020, 16, 2854–2861, doi:10.3762/bjoc.16.234

• structure and function studies of nucleic acids. Often, the effort to synthesize a specifically modified oligonucleotide is underestimated, since a wide spectrum of precursors and standard methodology is available. However, dependent on the specific synthetic aim, standard methods can fail or lead to

Synthesis and investigation of quadruplex-DNA-binding, 9-O-substituted berberine derivatives

• Jonas Becher,
• Daria V. Berdnikova,
• Heiko Ihmels and
• Christopher Stremmel

Beilstein J. Org. Chem. 2020, 16, 2795–2806, doi:10.3762/bjoc.16.230

• terminated with an N-benzylamide functionality to establish the attractive hydrogen bonding and π stacking with the thymidine residues in the loops in G4-DNA, so that this ligand binds with very high selectivity to the particular quadruplex-forming oligonucleotide J19 [49]. Overall, the above-mentioned

• rising concentration of the ligand and with increasing chain length n of the ligands 4a–e, as indicated by the shifts of the melting temperature of up to ΔTm = 12.9 °C (Table 1). In contrast, the oligonucleotide Fa2T is only stabilized to a negligible extent upon the association of the ligands 4a–e

• ). Furthermore, the selectivity of the ligand 4e toward quadruplex DNA in competition with duplex DNA was investigated by DNA denaturation experiments in the presence of an excess of the duplex-DNA forming oligonucleotide ds26. Under these conditions, the ligand 4e shows essentially the same stabilization as in

Naphthalene diimide–amino acid conjugates as novel fluorimetric and CD probes for differentiation between ds-DNA and ds-RNA

• Annike Weißenstein,
• Myroslav O. Vysotsky,
• Ivo Piantanida and
• Frank Würthner

Beilstein J. Org. Chem. 2020, 16, 2032–2045, doi:10.3762/bjoc.16.170

• polynucleotides we have chosen synthetic long ds-DNA: poly(dG-dC)2 and poly(dA-dT)2 and ds-RNA: poly(A)-poly(U), as well as mixed sequence ct-DNA (48% of G-C base pairs). The reason for the use of long polynucleotides was to avoid the physiologically non-relevant interactions with a short duplex oligonucleotide

• ). Schematic representation of the alignment of the intercalating 3a (left) and 3b (right) between the base pairs of the oligonucleotide. The complex with 3b was prepared analogously to the NDI analogue [53] by replacing the threading intercalator in PDB258D [54] with 3b, and performing MM2 minimisation in

A smart deoxyribozyme-based fluorescent sensor for in vitro detection of androgen receptor mRNA

• Ekaterina A. Bryushkova,
• Erik R. Gandalipov and
• Julia V. Nuzhina

Beilstein J. Org. Chem. 2020, 16, 1135–1141, doi:10.3762/bjoc.16.100

• receptor mRNA was developed. It consists of several functional modules including two deoxyribozymes 10–23, an RNA-dependent split malachite green aptamer, and an oligonucleotide platform. Deoxyribozymes specifically release a 27-nucleotide RNA fragment that is readily available for the interaction with the

• electrode, covered by immobilizing enzyme glucose oxidase, for the detection of glucose [1]. A biosensor means a small molecular device that traditionally consists of a bioreceptor (enzyme, cell, aptamer, oligonucleotide, antibody, and other) for the specific recognition of the target molecule and a

• deoxyribozymes 10–23 (Figure 1B) with different lengths of RNA-binding sites (Dz1 9 nt right/4 nt left and Dz2 9 nt right/8 nt left), which recognize 60-AR_RNA on both sides around the site of aptamer binding, (ii) split malachite green aptamer (Figure 1C), and (iii) an oligonucleotide platform (Figure 1A, T5

Photocontrolled DNA minor groove interactions of imidazole/pyrrole polyamides

• Sabrina Müller,
• Jannik Paulus,
• Jochen Mattay,
• Heiko Ihmels,
• Veronica I. Dodero and
• Norbert Sewald

Beilstein J. Org. Chem. 2020, 16, 60–70, doi:10.3762/bjoc.16.8

• ´-TTGTC-3´ for P1, 5´-TTGTT-3´ for P2, and 5´-TTGTCA-3´ for P3 (Scheme 1). Furthermore, as proof of concept, short hairpin oligonucleotide sequences were employed to investigate the interaction of the polyamides with dsDNA because six- and eight-membered polyamides can only bind in a 1:1 mode, and thus

• minimum at ≈250 nm were detected upon addition of polyamide. Considering that PAs do absorb light at wavelengths < 300 nm, the observed changes cannot be unambiguously attributed to the DNA because the ICD of the PA is combined with the intrinsic CD spectrum of the oligonucleotide. Nevertheless, the

• the ligands E-P1–E-P3 did not bind significantly to the oligonucleotide sequences D1–D3. However, considering their polyamide structure, they should at least exhibt a weak affinity toward double-stranded DNA. The fluorescent indicator displacement (FID) assay was performed with E-P1 as representative

In search of visible-light photoresponsive peptide nucleic acids (PNAs) for reversible control of DNA hybridization

• Lei Zhang,
• Greta Linden and
• Olalla Vázquez

Beilstein J. Org. Chem. 2019, 15, 2500–2508, doi:10.3762/bjoc.15.243

• tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore

• first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science. Keywords: azobenzene; hemithioindigo; peptide nucleic acid (PNA); photoswitch; visible-light irradiation; Introduction Light-driven control of oligonucleotide

• after the cleavage from the solid support. Among the initial studied photoswitchable PNAs, the PNA12(oF4Azo) (3) displayed the most promising properties as reversible modulator of oligonucleotide hybridization. Thus, it displayed the fastest reversible isomerization (≈2 s for cis → trans and ≈120 s for

Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries

• Xiao Xie,
• Michela Zuffo,
• Marie-Paule Teulade-Fichou and
• Anton Granzhan

Beilstein J. Org. Chem. 2019, 15, 1872–1889, doi:10.3762/bjoc.15.183

• 22 nt oligonucleotide. All samples were prepared in a K+-rich buffer solution (K-100, see Table 1 footnote). Thioflavin T (ThT), which is widely used for detection of G4 structures, was included for comparison. The fluorescence intensity was measured using a microplate reader. In order to screen a

• oligonucleotide, K-100 buffer). The data points corresponding to the oligonucleotides in the set are displayed in a 2D scatter plot (Figure 8), featuring normalized emission intensities of 1p and 18a dyes as x and y axes, respectively. Notably, the oligonucleotides appeared to be grouped in clusters broadly

• the existence of an equilibrium between a monomolecular antiparallel form and a bimolecular parallel one, affected by K+ and oligonucleotide concentration [86]. As already suggested, this oligonucleotide is probably present as a mixture of conformations in our working conditions [14]. It is thus

Tuning the stability of alkoxyisopropyl protection groups

• Zehong Liang,
• Henna Koivikko,
• Mikko Oivanen and
• Petri Heinonen

Beilstein J. Org. Chem. 2019, 15, 746–751, doi:10.3762/bjoc.15.70

• only the 2-methoxypropan-2-yl protecting group (MIP) has been adopted in use [2] and the alternative, 2-benzyloxypropan-2-yl, introduced by Mukaiyama in the 1980’s [6] has not gained popularity. The MIP group has been used, e.g., in solution-phase oligonucleotide synthesis for the primary 5’-hydroxy

• . The vinyl ether hydrolysis has earlier shown to be even a subject of general acid catalysis [13][14]. It is useful to compare the stabilities of the acetal protections to those of the 4,4’-dimethoxytrityl protecting group used in oligonucleotide synthesis. Directly comparable data are hard to find

• conditions typically applied in oligonucleotide synthesis, i.e., 3% dichloroacetic acid in acetonitrile or in methanol. In most cass the reactions were complete after a couple of minutes. With the trifluoroethyl acetal derivative 4e the reaction was slow enough that the half-life could be measured, and it

Aqueous olefin metathesis: recent developments and applications

• Valerio Sabatino and
• Thomas R. Ward

Beilstein J. Org. Chem. 2019, 15, 445–468, doi:10.3762/bjoc.15.39

• undergo Ru-promoted RCM and CM when the G-III catalyst is used under heterogeneous conditions (water/tert-butanol 3:2) with a large excess of Mg2+. Also in this case, the role of Mg2+ is to protect the oligonucleotide from Ru-induced decomposition by binding to the phosphate backbone. Table 12 summarizes

Synthesis, biophysical properties, and RNase H activity of 6’-difluoro[4.3.0]bicyclo-DNA

• Sibylle Frei,
• Adam K. Katolik and
• Christian J. Leumann

Beilstein J. Org. Chem. 2019, 15, 79–88, doi:10.3762/bjoc.15.9

• conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand. Keywords: DNA/RNA affinity; fluorinated cyclopropanes; fluorinated nucleic acids; RNase H activity; sugar modified nucleosides

• cleavage of the RNA strand of an AON/RNA hybrid structure by the endonuclease RNase H [9][18]. Both, the F–ANA and the F–RNA, are appealing modifications for several oligonucleotide-based silencing applications [8][19][20][21][22][23][24][25]. Also evaluated on their antisense properties were the 3

• intensities were changed, too. Furthermore, the intensity of the 210 nm peak was reduced. RNase H cleavage assay The most important requirement for an antisense oligonucleotide to induce RNase H activity lies in its DNA-like sugar conformations [49]. This is generally fulfilled by the 6’-diF-bc4,3-DNA as well

6’-Fluoro[4.3.0]bicyclo nucleic acid: synthesis, biophysical properties and molecular dynamics simulations

• Sibylle Frei,
• Andrei Istrate and
• Christian J. Leumann

Beilstein J. Org. Chem. 2018, 14, 3088–3097, doi:10.3762/bjoc.14.288

• = −1.5 to −3.7 °C). Molecular dynamics simulation on the nucleoside and oligonucleotide level revealed the preference of the C1’-exo/C2’-endo alignment of the furanose ring. Moreover, the simulation of duplexes with complementary RNA disclosed a DNA/RNA-type duplex structure suggesting that this

• RNase H1 which selectively cleaves the RNA strand of a DNA/RNA hybrid duplex [8]. To activate this process, fully modified DNA-like oligonucleotides (ONs) or gapmer AONs are used [9][10]. However, the widespread use of oligonucleotide-based therapeutics is limited by several factors amongst which the

• polarizability of the modified nucleotide, and therefore influences the metabolic stability, the membrane permeability, the RNA- and protein-binding affinity of the AON [25][26][27][28][29]. Over the last almost two decades, fluorinated oligonucleotide analogs like 2’-deoxy-2’-fluoro-RNA (F-RNA) [26][30][31], 2

Artificial bioconjugates with naturally occurring linkages: the use of phosphodiester

• Takao Shoji,
• Hiroki Fukutomi,
• Yohei Okada and
• Kazuhiro Chiba

Beilstein J. Org. Chem. 2018, 14, 1946–1955, doi:10.3762/bjoc.14.169

• bioactivities and/or functions, which has been one of the central topics in the field of chemical biology [16][17][18][19][20][21][22][23][24][25][26]. Since peptide synthesis and oligonucleotide synthesis require different chemistries, such conjugations are typically carried out in the latter stages of the

• linkages that can be formed between native functional groups would be safe alternatives, and are known as natural conjugates such as nucleopeptides and nucleolipids [49]. We have been developing alkyl chain soluble support (ACSS)-assisted liquid-phase methods, specifically for peptide and oligonucleotide

• supported reactants would be unique alternatives that allow the use of unactivated amino acids or nucleosides. In peptide synthesis, activation of the N-terminus is rather rare, except for some recent encouraging examples [62][63][64][65][66][67][68]; however, this is not the case for oligonucleotide

Phosphoramidite building blocks with protected nitroxides for the synthesis of spin-labeled DNA and RNA

• Timo Weinrich,
• Eva A. Jaumann,
• Ute M. Scheffer,
• Thomas F. Prisner and
• Michael W. Göbel

Beilstein J. Org. Chem. 2018, 14, 1563–1569, doi:10.3762/bjoc.14.133

• resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin–spin distances measured by PELDOR correspond well to the values obtained from molecular models. Keywords: EPR; oligonucleotide; PELDOR; photolabile protection; TEMPO

Oligonucleotide analogues with cationic backbone linkages

• Melissa Meng and
• Christian Ducho

Beilstein J. Org. Chem. 2018, 14, 1293–1308, doi:10.3762/bjoc.14.111

• motifs, and nucleosyl amino acid (NAA)-derived modifications. The synthesis and properties of the corresponding oligonucleotide analogues are described. Keywords: backbone modifications; cations; DNA; oligonucleotides; zwitterions; Introduction Oligonucleotides have the unique ability to bind

• a specific protein, which leads to effective (though reversible) and selective downregulation of the protein's activity. A third option for the biological action of oligonucleotide structures is the triggering of the RNA interference mechanism by double-stranded 'small interfering' RNA (siRNA

• aspired improvement of cellular uptake, fully cationic oligonucleotide analogues might also be attractive candidate structures, as indicated by the advantageous properties of cationic cell-penetrating peptides (CPPs) [38]. However, the design of modifications of type 1–6 precludes the preparation of fully