Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

A broadly applicable, robust, and rapid method for complete sequence confirmation of highly modified oligonucleotides containing a mixture of 2′‐deoxy, 2′‐fluoro, 2′‐o‐methyl, abasic and ribonucleotides is presented. The passenger (sense) and guide (antisense) strands from synthetic short interfering RNA duplexes (siRNA) were digested individually using both 5′‐ and 3′‐exonucleases and the resulting ladders were analyzed using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Conditions for enzymatic digestion and MALDI‐TOF mass analysis were investigated and optimized, and the digestion pattern and sequence coverage of each strand was discussed. Complete sequence confirmation for the antisense strands of four synthetic RNA duplexes was obtained, whereas a three‐base sequence gap in the 5′‐end was observed for all four sense strands. A general strategy is proposed for routine sequence confirmation of highly modified oligonucleotides, and the potential for complete automation of the method is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotide Duplexes (=p‐DNA Duplexes) as Substitutes of RNA Hairpin Structures

By automated synthesis, we prepared hybrid oligonucleotides consisting of covalently linked RNA and p‐DNA sequences (p‐DNA=3′‐deoxyribopyranose (4′→2′)‐oligonucleotides) (see Table 1). The pairing properties of corresponding hybrid duplexes, formed from fully complementary single strands were investigated. An uninterrupted π‐π‐stacking at the p‐DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4′‐p‐DNA‐2′→5′‐RNA‐3′ and 5′‐RNA‐2′→4′‐p‐DNA‐2′ phosphodiester linkage, respectively (see Fig. 4). The RNA 2′‐phosphoramidites 9 – 12 , required for the formation of the RNA‐2′→4′‐p‐DNA phosphodiester linkage were synthesized from the corresponding, 3′‐O‐tom‐protected ribonucleosides (tom=[(triisopropylsilyl)oxy]methyl; Scheme 1). Analogues of the flavin mononucleotide (=FMN) binding aptamer 22 and the hammerhead ribozyme 25 were prepared. Each of these analogues consisted of two p‐DNA/RNA hybrid single strands with complementary p‐DNA sequences, designed to substitute stem/loop and stem motifs within the parent compounds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p‐DNA/RNA hybrid sequences resulted in the formation of the fully functional analogues 23 ⋅ 24 and 27 ⋅ 28 of the FMN‐binding aptamer and of the hammerhead ribozyme, respectively.     

Molecular‐Dynamics Studies of Single‐Stranded Hexitol,Altritol, Mannitol,and Ribose Nucleic Acids (HNA,MNA, ANA,and RNA,Resp.) and of the Stability of HNA⋅RNA,ANA⋅RNA,and MNA⋅RNA Duplexes

The influence of the orientation of a 3′‐OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular‐dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long‐range electrostatic interactions. An equatorial orientation of the 3′‐OH group in the single‐stranded D ‐mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H‐bonds between the 3′‐OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′‐OH group in the D ‐altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single‐stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A‐RNA, with only minor changes in groove width, helical curvature, and H‐bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D ‐hexitol nucleic acid without 3′‐OH group) can be explained by differences in restricted movements, H‐bonds, and solvation effects.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

Hairpin DNA‐Dependent Click Conjugation of Oligonucleotides for Electrochemical Monitoring of Copper(II)

A new electrochemical sensing platform was designed for sensitive detection of copper(II) (Cu²⁺) based on click conjugation of two short oligonucleotides by using methylene blue‐functionalized hairpin DNA as the template. The analyte (Cu²⁺) was in situ reduced to Cu⁺ by sodium ascorbate, which catalyzed the click conjugation between two single‐stranded oligonucleotides one was labelled with a 5′‐alkyne and the other with 3′‐azide group via the Cu⁺‐catalyzed azide‐alkyne cycloaddition. The newly formed long‐chain oligonucleotide induced the conformational change of hairpin DNA to open the hairpin, resulting in methylene blue far away from the electrode for the decrease of redox current. Under optimal conditions, the decrease in the electronic signal was directly proportional to target Cu²⁺ concentration, and allowed the detection of Cu²⁺ at a concentration as low as 1.23 nM. Our strategy also displayed high selectivity for Cu²⁺ against other metal ions owing to the highly specific Cu⁺‐catalyzed click chemistry reaction, and was applicable for monitoring of Cu²⁺ in drinking water with satisfactory results.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

Factors Affecting DNA–DNA Interstrand Cross‐Links in the Antiparallel 3′–3′ Sense: A Comparison with the 5′–5′ Directional Isomer

Reported herein is a study of the unusual 3′–3′ 1,4‐GG interstrand cross‐link (IXL) formation in duplex DNA by a series of polynuclear platinum anticancer complexes. To examine the effect of possible preassociation through charge and hydrogen‐bonding effects the closely related compounds [{trans‐PtCl(NH₃)₂}₂(μ‐trans‐Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂)]⁴⁺ (BBR3464, 1 ), [{trans‐PtCl(NH₃)₂}₂(μ‐NH₂(CH₂)₆NH₂)]²⁺ (BBR3005, 2 ), [{trans‐PtCl(NH₃)₂}₂(μ‐H₂N(CH₂)₃NH₂(CH₂)₄)]³⁺ (BBR3571, 3 ) and [{trans‐PtCl(NH₃)₂}₂{μ‐H₂N(CH₂)₃‐N(COCF₃)(CH₂)₄}]²⁺ (BBR3571‐COCF₃, 4 ) were studied. Two different molecular biology approaches were used to investigate the effect of DNA template upon IXL formation in synthetic 20‐base‐pair duplexes. In the “hybridisation directed” method the monofunctionally adducted top strands were hybridised with their complementary 5′‐end labelled strands; after 24 h the efficiency of interstrand cross‐linking in the 5′–5′ direction was slightly higher than in the 3′–3′ direction. The second method involved “postsynthetic modification” of the intact duplex; significantly less cross‐linking was observed, but again a slight preference for the 5′–5′ duplex was present. 2D [¹H, ¹⁵N] HSQC NMR spectroscopy studies of the reaction of [¹⁵N]‐ 1 with the sequence 5′‐d{TATACATGTATA}₂ allowed direct comparison of the stepwise formation of the 3′–3′ IXL with the previously studied 5′–5′ IXL on the analogous sequence 5′‐d(ATATGTACATAT)₂. Whereas the preassociation and aquation steps were similar, differences were evident at the monofunctional binding step. The reaction did not yield a single distinct 3′–3′ 1,4‐GG IXL, but numerous cross‐linked adducts formed. Similar results were found for the reaction with the dinuclear [¹⁵N]‐ 2 . Molecular dynamics simulations for the 3′–3′ IXLs formed by both 1 and 2 showed a highly distorted structure with evident fraying of the end base pairs and considerable widening of the minor groove.     

Increased Affinity of 2′‐O‐(2‐Methoxyethyl)‐Modified Oligonucleotides to RNA through Conjugation of Spermine at Cytidines

Structural modification at the 2′‐O‐position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotides that have been tested in clinical trials. Yet, the search for new improved modifications continues in a quest for increased oligonucleotide potency, improved transport in vivo and favorable metabolism. Recently, we described how the conjugation of spermine groups to pyrimidines in oligonucleotides vastly increases their affinity for complementary RNAs through accelerated binding kinetics. Here we describe how spermines can be linked to the exocyclic amino groups of cytidines in MOE‐oligonucleotides employing a straightforward ‘convertible nucleoside approach’ during solid phase synthesis. Singly‐ or doubly‐modified oligonucleotides show greatly enhanced affinity for complementary RNA, with potential for a new generation of MOE‐based oligonucleotide drugs.     

Oligodeoxynucleotides Containing 2′‐Deoxy‐1‐methyladenosine and Dimroth Rearrangement

2′‐Deoxy‐1‐methyladenosine was incorporated into synthetic oligonucleotides by phosphoramidite chemistry. Chloroacetyl protecting group and controlled anhydrous deprotection conditions were used to avoid Dimroth rearrangement. Hybridization studies of intramolecular duplexes showed that introduction of a modified residue into the loop region of the oligonucleotide hairpin increases the melting temperature. It was shown that modified oligonucleotides may be easily transformed into oligonucleotides containing 2′‐deoxy‐N⁶‐methyladenosine.     

Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MSE analysis of RNA oligonucleotides

Fast and efficient ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence‐related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS^E (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS^E technique is as efficient as the traditional MS/MS method, yet MS^E is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2′‐ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS^E approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS^E. Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time‐of‐flight (QTOF) MS/MS and MS^E methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

A Novel Synthesis of the Macrocyclic Spermidine Alkaloid (+)‐(S)‐Dihydroperiphylline

A novel, short, and highly stereoselective synthesis of the macrocyclic spermidine alkaloid (+)‐(S)‐dihydroperiphylline ( 15 ) is described. The key synthetic steps were the stereoselective addition of the chiral amine 1 to the cinnamate 2 and cyclization of the bis[toluene‐4‐sulfonamide] precursor 12 in the presence of Cs₂CO₃ as a template. Unambiguous assignments of the signals in both the ¹H‐ and ¹³C‐NMR spectra of 15 were achieved by 2D NMR spectra.     

Synthesis and Base Pairing Properties of 1′,5′‐Anhydro‐L‐Hexitol Nucleic Acids (L‐HNA)

Oligonucleotides composed of 1′,5′‐anhydro‐arabino‐hexitol nucleosides belonging to the L series (L ‐HNA) were prepared and preliminarily studied as a novel potential base‐pairing system. Synthesis of enantiopure L ‐hexitol nucleotide monomers equipped with a 2′‐(N⁶‐benzoyladenin‐9‐yl) or a 2′‐(thymin‐1‐yl) moiety was carried out by a de novo approach based on a domino reaction as key step. The L oligonucleotide analogues were evaluated in duplex formation with natural complements as well as with unnatural sugar‐modified oligonucleotides. In many cases stable homo‐ and heterochiral associations were found. Besides T_m measurements, detection of heterochiral complexes was unambiguously confirmed by LC‐MS studies. Interestingly, circular dichroism measurements of the most stable duplexes suggested that L ‐HNA form left‐handed helices with both D and L oligonucleotides.     

5‐Aza‐7‐deazaguanine DNA: Recognition and Strand Orientation of Oligonucleotides Incorporating Anomeric Imidazo[1,2‐a]‐1,3,5‐triazine Nucleosides

The base‐pairing properties of oligonucleotides containing the anomeric 5‐aza‐7‐deazaguanine 2′‐deoxyribonucleosides 1 and 5 are described. The oligonucleotides were prepared by solid‐phase synthesis, employing phosphoramidite or phosphonate chemistry. Stable `purine'⋅purine duplexes with antiparallel (aps) chain orientation are formed, when the α‐D ‐anomer 5 alternates with the β‐D ‐anomeric 2′‐deoxyguanosine ( 2 ) within the same oligonucleotide chain. Parallel (ps) oligonucleotide duplexes are observed, when the β‐D anomer 1 alternates with 2 . A renewed reversal of the chain orientation (ps→aps) occurs when compound 1 pairs with 2′‐deoxyisoguanosine ( 6 ). In all cases, it is unnecessary to change the orientation within a single strand when α‐D units alternate with their β‐D counterparts. Heterochiral base pairs of 5 (α‐D ) with 2′‐deoxyisoguanosine (β‐D ) are well accommodated in duplexes with random base composition and parallel chain orientation. Base pairs of 5 (α‐D ) with 2′‐deoxyguanosine (β‐D ) destabilize duplexes with antiparallel chains.     

Synthesis and Triple‐Helix‐Stabilization Properties of Branched Oligonucleotides Carrying 8‐Aminoadenine Moieties

The synthesis of several branched oligonucleotides, i.e., of the parallel hairpins 5 – 8 and the Y‐shaped 9 is described, together with their use in the formation of pyrimidine?pyrimidine?purine triple helices. Special attention was paid to the optimization of the assembly of the second strand from asymmetric branching molecules. The presence of 8‐aminoadenine moieties in the Watson? Crick purine strand and 2′‐O‐methyl‐RNA in the Hoogsteen pyrimidine strand produced strong stabilization of the triplex.     

Duplex‐forming Oligonucleotide of Triazole‐linked RNA

An oligonucleotide of triazole‐linked RNA (^TLRNA) was synthesized by performing consecutive copper‐catalyzed azide‐alkyne cycloaddition reactions for elongation. The reaction conditions that had been optimized for the synthesis of 3‐mer ^TLRNA were found to be inappropriate for longer oligonucleotides, and the conditions were reoptimized for the solid‐phase synthesis of an 11‐mer ^TLRNA oligonucleotide. Duplex formation of the 11‐mer ^TLRNA oligonucleotide was examined with the complementary oligonucleotide of natural RNA to reveal the effects of the 2′‐OH groups on the duplex stability.     

Facile Synthesis of Diastereoisomerically and Optically Pure 2‐Substituted Hexahydro‐1H‐pyrrolizin‐3‐ones

We report a short synthetic route that provides optically active 2‐substituted hexahydro‐1H‐pyrrolizin‐3‐ones in four steps from commercially available Boc (tert‐but(oxy)carbonyl))‐protected proline. Diastereoisomers (−)‐ 11 and (−)‐ 12 were assembled from the proline‐derived aldehyde (−)‐ 8 and ylide 9 via a Wittig reaction and subsequent catalytic hydrogenation (Scheme 3). Cleavage of the Boc protecting group under acidic conditions, followed by intramolecular cyclization, afforded the desired hexahydro‐1H‐pyrrolizinones (−)‐ 1 and (+)‐ 13 . Applying the same protocol to ylide 19 afforded hexahydro‐1H‐pyrrolizinones (−)‐ 25 and (−)‐ 26 (Scheme 5). The absolute configuration of the target compounds was determined by a combination of NMR studies (Figs. 1 and 2) and X‐ray crystallographic analysis (Fig. 3).     

Ein neuer Zugang zu 2′‐O‐(2‐Methoxyethyl)ribonucleosiden ausgehend von D‐Glucose

A New Access to 2′‐ O ‐(2‐Methoxyethyl)ribonucleosides Starting from D ‐Glucose A new synthesis of 2′‐O‐(2‐methoxyethyl)ribonucleosides, building blocks for second‐generation antisense oligonucleotides, starting from D ‐glucose is presented. The key‐step is the transformation of 3‐O‐methoxyethylallofuranose to 2‐O‐(2‐methoxyethyl)ribose by NaIO₄ oxidation. Together with the 4′‐phenylbenzoyl protecting group, which results in crystalline intermediates, this synthesis provides an easy and cheap access to 2′‐O‐(2‐methoxyethyl)‐substituted ribonucleosides.     

Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.