Stability leads to selection: Cationic appending groups on a triplex-forming oligonucleotide (TFO) designed to bind a DNA target can be rapidly selected from a small library of various amines and polyamines for their capacity to stabilize triple-helix formation. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Oligonucleotides complementary to RNA sequences interact poorly with folded target regions. In vitro selection of oligonucleotides carried out against RNA structures have led to aptamers that frequently differ from antisense sequences, but rather take advantage of non-double-stranded peculiarities of the target. Studies along this line provide information about tertiary RNA architectures as well as their interaction with ligand of interest. We describe here a genomic SELEX approach and its application to the recognition of stem-loop structures prone to the formation of kissing complexes. We also provide technical details for running a procedure termed 2D-SELEX that takes advantage of both in vitro selection and dynamic combinatorial chemistry. This allows selecting aptamer derivatives containing modified nucleotides that cannot be incorporated by polymerases. Last we present in vitro transcription conditions under which large amounts of RNA, suitable for NMR structural studies, can be obtained. These different aspects of the SELEX technology have been applied to the trans-activating responsive element of the human immunodeficiency virus type 1, which is crucial for the transcription of the retroviral genome.

SELEX (for Systematic Evolution of Ligands by Exponential enrichment) has proven to be extraordinarily powerful for the isolation of DNA or RNA aptamers that bind with high affinity and specificity to a wide range of molecular targets. However, the modest chemical functionality of nucleic acids poses some limits on the versatility of aptamers as binders and catalysts. To further improve the properties of aptamers, additional chemical diversity must be introduced. The design of chemical modifications is not a trivial task. Recently, dynamic combinatorial chemistry (DCC) has been introduced as an alternative to traditional combinatorial chemistry. DCC employs equilibrium shifting to effect molecular evolution of a dynamic combinatorial library of molecules. Herein, we describe an original process that combines DCC and SELEX for the in vitro selection of modified aptamers which are conjugated to chemically diverse small-molecules. Its successful application for the selection of small-molecule conjugated RNA aptamers that bind tightly to the transactivation-response (TAR) element of HIV-1 is presented.

During the synthesis of oligonucleotides by the standard phosphoramidite method using 2'-deoxycytidine- derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3'-terminal 2'-deoxycytidine. Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.

A dynamic library of 15 mono- and bi-conjugated oligonucleotides was generated from a pool of three aldehydes and an oligonucleotide bearing two reactive amino groups. Addition of complementary target to the equilibrating mixture of imines resulted in selective amplification of one conjugate. UV-melting experiments confirmed that it was the best ligand among those that were tested. This study emphasizes that dynamic combinatorial chemistry can be used to simultaneously identify the type and the location of appended residues for stabilizing oligonucleotide complexes. (C) 2004 Elsevier Ltd. All rights reserved.

The p-complementary hexamer, p-d[GTACGC], t o the a s e q u e n c e , a-d[CATGCG], was s y n t h e s i z e d by the p h o s p h o t r i e s t e r method. The nonexchangeable proton a s s i g n n e n t s were obta ined u s i n g 1Dand 2D-NMR t e c h n i q u e s , i n c l u d i n g NOE, COSY and NOESY. The p s t rand e x i s t s as a random coi l at 21°C; however, at 4°C, i t forms an antiparallel self-recognition duplex annealing at positions 1-4. The p-strand was annealed to the oc-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in HjOA^O solution at 21°C. 1D-N0E experiments of the a, p duplex d[ a(CATGCG)•p-(GTACGC)] reveal that (i) i t exists in aqueous solution in a conformation that belongs to the B family, ( i i ) i t i s 70±10% right-handed, ( i i i ) the sugar-base orientations of the p-strand are anti, and the deoxyribose units exist predominantly in the 2*-endo-3'-exo conformation. NOE measurements of the imino proton signals in the a,p duplex reveal that the duplex exhibits parallel polarity. INTRODUCTION The regulation of gene expression in both procaryotes and eucaryotes requires the specific recognition of single-stranded or double-stranded nucleic acid base sequences by sequence specific proteins, repressor RNA's or synthetic probes (1-3) . The abi l i ty to design sequence specific agents to serve as vectors for the delivery of UNA effectors could have a major impact in providing new probes for obtaining deeper knowledge of gene expression and control. A number of promising approaches to the latter problem include the use of natural (4-5), and rationally altered sequence specific oligopeptides (6), and p-ollgonucleotides (7-11) as vectors. A major problem in using natural p-oligonucleotides as vectors i s that they are subject to nuclease degradation in the ce l l (12-13). Accordingly, we are exploring the potential of the unnatural a-oligomers as vectors. Recent studies have shown that a-oligonucleotides are more resistant towards hydrolysis than their p-cogeners. For example, the unnatural hexadeoxyribonucleotide a-(d(CpApTpGpCpG)) remains intact under conditions where the natural corresponding p-hexamer was completely degraded by endonuclease SI © IRL Pnw Limited, Oxford, England. 7 0 2 7 Downloaded from https://academic.oup.com/nar/article-abstract/15/17/7027/1373326/DNA-V-Parallel-annealing-handedness-and by guest on 15 September 2017 Nucleic Acids Research and calf spleen phosphodiesterase (14). In 1973, using Drieding stereomodels, U. Sequin (15) predicted that an a-atrand should form a helix duplex with a complementary Bor a-strand by base pairing and the two strands should exhibit parallel and antiparallel polarity, respectively. We recently described the synthesis and high f ie ld H NKR characterization of o-[d(CpCpTpTpCpC)] and demonstrated that i t i s capable of annealing to i t s B—complement (16). That prototype a-sequence, selected primarily for reasons of synthetic convenience, was not appropriate for determining such crucial properties as the polarity of annealing (due to the symmetry of the oe-oligomer), the extent and nature of self-recognition, and the conformation of annealed structures. Accordingly, we have recently reported the synthesis (14) and characterization by high field H NMR of the hexamer o-td(CpApTpGpCpG)] (17). The study revealed antiparallel self-recognition and duplex annealing at positions 1-4 for a-[d(CATGCG)], and temperature variation of the imino H NMR signals suggests that the hydrogen bonding in self-recognition i s comparable in strength with that in p-DNA duplex, and NOE data are in accord with Watson-Crick base pairing (17). Therefore, the next requirement is to determine the structure and conformation of o-[d(CATGCG)] annealed to i t s B-complement, nanely B-[ d (GpTpApCpGpC) ] . He report the H NMR characterisation of the ^-complementary hexamer B-[d(GTACGC)1, and the corresponding annealed duplex d[o-(C1A2T3G4C5G6) •p-(G7TgAgC10G1 -|C12) ] , as well as deduction of the polarity, handedness and conformational features of the lat ter . MATERIALS AND METHODS Synthesis Both hexamers a-[d(CpApTpGpCpG)] and B[d(GpTpApCpGpC) ] were synthes ized by the phosphotr ies ter method in s o l u t i o n ( 1 4 , 1 6 ) . NMR Spectroscopy The NMR of B-d[GTACGC] was prepared by d i s s o l v i n g 12.7 mg of the hexamer in a 99.8% D20 (Aldrich) so lu t ion conta in ing 40 mM potassium phosphate (pH 7 .2) and 20 mM sodiun c h l o r i d e . The s o l u t i o n was lyoph i l i zed twice from 99.8% DjO (Aldrich) and once from 99.996% D20, and f i n a l l y made up to 0 .4 mL with 99.996% DjO (Aldr ich) . The double stranded sample d[a-(CATGCG)•B-(GTACGC)1 was prepared by d i s s o l v i n g 12.7 tag of each strand in a 20 raM sodium c h l o r i d e , 40 mM potassium phosphate (pH 7.2) s o l u t i o n (0 .4 mL). The s o l u t i o n was maintained

In vitro selection or systematic evolution of ligands by exponential enrichment is a combinatorial procedure that allows the identification of oligonucleotides showing properties of interest-so-called aptamers-through iterative selection/amplification rounds. Libraries containing as many as 1014 different sequences can be screened against a wide range of molecules. Ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or chemically modified aptamers generally display high affinity and exquisite specificity of interaction with the target. Aptamers show a promising potential for diagnostic and therapeutic purposes. We describe here methods successfully used in our laboratory for the selection of RNA or DNA aptamers against an RNA structure (the transactivation response element of HIV-1) and a protein (the human ribonuclease H1).

Der beste kovalent gebundene Ligand, der einen Oligonucleotidkomplex stabilisiert, wird mithilfe eines dynamischen kombinatorischen Ansatzes schnell identifiziert, bei dem dieser Ligand amplifiziert wird (siehe schematische Darstellung). Sowohl ein DNA-Duplex als auch ein RNA-„Kiss“-Komplex wurden untersucht. Schmelztemperaturexperimente bestätigten, dass die amplifizierten Spezies die Komplexe stabilisieren. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2004/z54041_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Monomeric units of nucleic acids DN A or RN A are characterized by a β configuration at the anomeric center of the sugar moiety (Fig. 1). Natural constitutive nucleosides of this configuration are readily commercially available, and from such starting building blocks, numerous phosphate-backbone-modified oligonucleotides have been proposed, the most well known being the methylphosphonate and the phosphorothioate series (). Open image in new window Fig. 1. Monomeric structure of oligonucleotides.

The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2'-deoxy-L-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central L2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking D4-sugar portions adopt the known Z left-handed conformation. The resulting Z4-B2*-Z4 structure (left-handed helix) is the reverse of that of B4-Z2*-B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base-base stackings and base-sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4-B2*-Z4 (respectively B4-Z2*-B4) helix displays a Tm (approximately 65 degrees C) that is only 5 degrees C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.

From propyne and 5-iodo-α,2-deoxyuridine, obtained by glycosylation, 5-propynyl-α,2′-deoxyuridine was synthesized following the procedure of Hobbs. One part was then transformed through displacement of its C4-triazolo derivative with ammonia into 5-propynyl-α,2′-deoxycytidine derivative. Finally, the corresponding α 5-propynyl nucleoside phosphoramidites were prepared, and 5-propynyl-α oligonucleotides (12-mer) with either phosphodiester and phosphorothioate were synthesized. The melting temperatures showed that duplexes with complementary DNA are stabilized between 0.65 and 1.2°C/mod, and duplexes with RNA are stabilized between 1.2 and 1.4°C/mod.

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Consequences of masking the phosphate functions of nucleic acid derivatives by enzyme labile bioreversible protecting groups are discussed.

The potential use of alpha-beta-anomeric duplex oligonucleotides to inhibit transcription factor activity by the decoy approach is investigated in this report. Indeed, several alpha-beta-anomeric heteroduplexes display a sequence-specific interaction with the p50 subunit of the transcription factor NF kappa B. Used in a decoy approach, these duplexes interact strongly enough with this transcription factor to modulate the expression of a reporter gene, under the control of NF kappa B. However, all the alpha-beta-anomeric heteroduplexes do not interact with the p50 subunit; the sequence of the chirally natural beta-anomeric strand may explain the different recognition properties of the protein. The analysis of the appropriate beta-anomeric sequences is consistent with a preferential interaction of the p50 subunit with one strand of double-stranded DNA.

Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected human CEM cells. Encapsulation in antibody-targeted liposomes (immunoliposomes) permitted intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. Our results are consistent with four mechanisms of antiviral activity for these antisense oligonucleotides: (i) interference with virus-mediated cell fusion by free but not liposome-encapsulated phosphorothioate oligonucleotides of any sequence; (ii) interference with reverse transcription in a sequence non-specific manner by phosphorothioate oligonucleotides in alpha and beta configurations; (iii) interference with viral reverse transcription in a sequence-specific and RNase-H-independent manner by alpha and beta phosphodiester oligonucleotides; (iv) interference with viral mRNA in a sequence-specific and RNase-H-dependent manner by beta-phosphorothioate oligonucleotides.

Two-dimensional NMR methods were used to model the possible solution structure of an intercalative complex of 9-aminoellipticine (Aell), a polycyclic pyridocarbazolamine, covalently bound to an apurinic ring-opened deoxyribose site of a duplex DNA fragment in the reduced Schiff base form. The required oligonucleotide single strand containing covalently attached aminoellipticine was obtained by reductive amination in the presence of sodium cyanoborohydride. The combined NMR-energy minimization methods were employed to refine the model structures of two distinct forms, intrahelical and extrahelical, of a control 9-mer duplex DNA, d(CGTG.dr.GTGC).d(GCACTCACG), which contains an apurinic site positioned opposite a dT residue on the complementary strand. The model structure of an aminoellipticine conjugate with the same DNA sequence, derivatized via the aforementioned covalent attachment, was also obtained by incorporating intermolecular drug-DNA and intra- and internucleotide NOE-derived proton-proton distance estimates as restraints in energy minimization routines. The indole ring system of aminoellipticine, which is inserted at the apurinic site, intercalates between and is parallel to flanking GC base pairs. The pyridinic ring of aminoellipticine, in protonated form, also stacks between cytidine and thymidine bases on the complementary strand, which is consistent with the observation that the normal sequential NOE connectivity at the 5'-C13-T14 step is broken and indeed diverted through the ellipticine moiety, e.g., C13-Aell-T14 connectivities through the Aell-H4/C5Me protons. Interestingly, the partial stacking of the pyridinic ring is observed only between the 5'-CT step vs an adjacent 5'-TC step, owing to inherently weak stacking interactions associated with the former. In the absence of any potential groups that can participate in electrostatic or hydrogen-bonding interactions with the nucleic acid, pi-pi stacking and hydrophobic contacts at the intercalation site appear to be the important factors in determining stability and conformation of the aminoellipticine-DNA conjugate. Stacking interactions in such a bistranded intercalative complexation of aminoellipticine apparently govern the formation of a single intrahelical form of a right-handed B-type DNA duplex. The overall structural features lead us to propose working models for an enzyme-like DNA cleavage activity of 9-aminoellipticine and the observed inhibition of the AP endonuclease-dependent DNA excision-repair pathway.