structural biology of small rna-mediated gene regulation abstracts - tuesday.pdf · tuesday, 7th...

Tuesday, 7th August 8.30 am

STRUCTURAL BIOLOGY OF SMALL RNA-MEDIATED GENE REGULATION

Dinshaw J. Patel Abby Rockefeller Mauze Chair in Experimental Therapeutics, Structural Biology Program, Memorial Sloan-Kettering

Cancer Center, 1275 York Avenue, New York NY-10065, USA

* Correspondence to: [email protected]

ABSTRACT

The lecture will focus on the structural biology of riboswitches, mRNA elements consisting of a sensing domain and an expression platform, that undergo conformational changes on metabolite binding, and

utilize on-off switches to control gene expression. This segment will be followed by our recent research on the structural biology of Argonaute and Dicer proteins and emerging mechanistic insights into cleavage events associated with RNA silencing.


STRUCTURAL BASIS OF LARIAT RNA RECOGNITION BY THE INTRON DEBRANCHING ENZYME, DBR1

Eric Montemayor1*, Adam Katolik2, Alexander Taylor1, Jonathan Schuermann3, Joshua Combs4, Richard Johnsson2, Stephen Holloway1, Masad J. Damha2, Scott Stevens4, P. John Hart1

1University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio TX 78229, 2McGill University, 801 Sherbrooke St. W. Montréal, Québec H3A 2K6, 3Cornell University, Ithaca, NY 14853, USA 4University of Texas,

Austin, TX 78712, USA. * Correspondence to: [email protected]

ABSTRACT

Here we present the first structures of the intron debranching enzyme Dbr1. The structures demonstrate how Dbr1 is capable of specifically recognizing the branchpoint structure within lariat RNA, and allow formulation of mechanistic hypothesis regarding hydrolysis of the unique 2�,5�-phosphodiester bond contained within lariat RNA.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

The spliceosome removes introns from pre-mRNA in the form of a lariat that contains an unusual 2�,5�-phosphodiester linkage. This linkage must be hydrolyzed by the intron debranching enzyme (Dbr1)1 before a spliced intron can be metabolized or processed into essential cellular factors such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons2 and HIV-13, ostensibly through a transient 2�,5�-phosphodiester linkage that has been proposed to facilitate the strand-transfer reaction of reverse transcription. Despite extensive biochemical characterization over several decades4-7, the exact enzyme mechanism and structural basis of lariat RNA recognition by Dbr1 has remained elusive. Here, we describe the first structures of Dbr1, in complex with several RNA compounds that mimic the branchpoint structure in lariat RNA. The structures demonstrate how Dbr1’s catalytic machinery is compatible with the 2�,5�-phosphodiester linkage and not the far more abundant 3�,5�-phosphodiester linkage. A combination of cell-based functional assays, in vitro activity assays, inductively coupled plasma mass spectrometry (ICP-MS) and X-ray anomalous diffraction methods were used to derive an enzyme mechanism for Dbr1. The proposed mechanism is novel in that it involves a dinuclear metal-binding center with functional alternation between single and double metal ion configurations. These findings provide a framework for understanding the role of Dbr1 in retrotransposon and retrovirus replication, and draw further attention to the potential evolutionary relationship between retrotransposons, retroviruses and pre-mRNA splicing.

REFERENCES

1. Ruskin, B. & Green, M.R. Science, 1985, 229, 135-140.

2. Cheng, Z. & Menees, T.M., Science, 2004, 303, 240-243.

3. Ye, Y., De Leon, J., Yokoyama, N., Naidu, Y. & Camerini, D., Retrovirology, 2005, 2, 63.

4. Khalid, M.F., Damha, M.J., Shuman, S. & Schwer, B. Nucleic Acids Res., 2005, 33, 6349-6360.

5. Ooi, S.L. et al. Methods Enzymol., 2001, 342, 233-248.

6. Nam, K. et al. J. Biol. Chem., 1994, 269, 20613-20621.

7. Arenas, J. & Hurwitz, J. J. Biol. Chem., 1987, 262, 4274-4279.

Figure 1. Molecular basis for how Dbr1 specifically hydrolyzes the 2�,5�-phosphodiester linkage in lariat RNA. The putative nucleophilic water and leaving group O2’ oxygen are shown as red spheres, while the active site metal ion is shown as a grey sphere. a) Proposed binding mechanism for 2�,5�-phosphodiester linkages. In this structure, the nucleophilic water is aligned to attack the phosphodiester bond, while His91 is positioned to act as a catalytic acid in protonating the leaving group oxygen. b)The structure of Dbr1 in complex with a 3�,5�-phosphodiester linkage, which is not hydrolyzed by the enzyme. Rotation of the ribose is necessary for this phosphodiester to be placed into the enzyme active site, causing a steric clash between the free 2�hydroxyl and His91 that precludes the latter from acting as a catalytic acid, thereby inactivating the enzyme.


SIRNA ACTIVITY, STABILITY AND 3D-STRUCTURAL PROPERTIES OF GNA-MODIFIED RNA

Martin Egli,1* Pradeep S. Pallan,1 Klaus Charisse2 and Muthiah Manoharan2

1Dept.of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA and 2Alnylam Pharmaceuticals Inc., 300 Third Street, Cambridge, MA 02142, USA. * Correspondence to: Email address [email protected]

ABSTRACT

Glycol nucleic acid (GNA) contains a chiral, acyclic three-carbon propylene glycol inter-nucleoside moiety. Despite the shorter backbone, S-GNA oligonucleotides form stable duplexes with RNA. We have investigated the RNA affinity, nuclease resistance, in vitro siRNA activity and 3D-structure of RNA oligonucleotides with incorporated S- or R-GNA nucleotides.

BACKGROUND AND RESULTS

Glycol nucleic acid (GNA; Figure 1) is arguably the simplest artificial nucleic acid pairing system with a phosphodiester backbone [1, 2]. Despite its acyclic backbone GNA is capable of stable self-pairing and the S-GNA enantiomer cross-pairs with RNA. Neither all-S- nor all-R-GNA oligonucleotides exhibit stable pairing with DNA.

Meggers and coworkers reported an initial crystal structure of an S-GNA octamer duplex whose right-handed geometry differed substantially from that of a canonical A-RNA duplex [3]. The duplex featured 16 base pairs per turn (average twist 23°) and a helical rise of 3.8 Å, with an average distance between adjacent phosphates of 5.4 Å. Despite the significant unwinding compared with A- and B-form duplexes, the strongly negative backbone inclination [4] and the short spacing between intra-strand phosphates were reminiscent of RNA. A second crystal structure of S-GNA provided evidence that the analogue exhibits considerable variations in its geometry [5]. Thus, this duplex displayed a compressed helical rise (2.6 Å) and twisting more like B-DNA (35.7°; 10 residues per turn), whereas average slide (-3.4 Å) and average intra-strand

phosphate-phosphate distance were virtually the same as in the structure determined earlier. The similar geometries help explain the pairing between S-GNA and RNA. We hypothesize that S-GNA, similar to DNA, is able to adapt to the conformational constraints of RNA and that the geometry of the GNA:RNA hybrid duplex resembles the A-form. Because R-GNA is left-handed and has a positive backbone-base inclination it is unable to pair with right-handed, negatively inclined RNA.

The surprisingly high stability of GNA duplexes is likely the result of extensive inter-strand base stacking and favorable, hydrophobic intra-strand contacts between nucleobase and backbone atoms [3]. It is also consistent with a significantly lower entropic penalty for duplex formation compared with DNA [6]. Given the lack of a cyclic sugar in the GNA backbone, this increased conformational preorganization of single strands is rather unexpected.

So far, the available information on the pairing and 3D-structure of GNA is limited to homo-oligonucleotides. To explore the effects of incorporation of modified nucleotides on RNAi activity [7] and RNA affinity, we produced siRNA duplexes containing S- or R-GNA base pairs. The presentation will provide a summary of the thermodynamic stability, nuclease resistance, in vitro activity and crystal structure of GNA-modified RNAs. Supported by the US NIH, R01 GM055237; Lab URL http://structbio.vanderbilt.edu/~eglim/

REFERENCES 1. Zhang, L., Peritz, A., Meggers, E. J. Am. Chem. Soc.

2005, 127, 4174-4175. 2. Meggers, E., Zhang, L. Acc. Chem. Res. 2010, 43,

1092-1102. 3. Schlegel, M.K., Essen L.-O., Meggers, E. J. Am. Chem.

Soc. 2008, 130, 8158-8159. 4. Pallan, P.S., Lubini, P., Bolli, M., Egli, M. Nucleic

Acids Res 2007, 35, 6611-6624. 5. Schlegel, M.K., Essen, L.-O., Meggers, E. Chem.

Comm. 2010, 1094-1096. 6. Schlegel, M.K., Xie, X., Zhang, L., Meggers, E. Angew.

Chem. Int. Ed. 2009, 48, 960-963. 7. Adepalli, H., Meena, Peng, C.G., Wang, G., Fan, Y.,

Charisse, K., Jayaprakash, K.N., Rajeev, K.G., Pandey, R.K., Lavine, G., Zhang, L., Jahn-Hofmann, K., Hadwiger, P., Manoharan, M., Maier, M.A. Nucleic Acids Res. 2010, 38, 7320-7331.

Figure 1. Structures of S-GNA and RNA.


Figure 2. Effects of oxidation on unfolding.

SINGLE-MOLECULE STUDIES OF OXIDATIVE DAMAGE IN TELOMERIC G QUADRUPLEXES

Aaron M. Fleming, Na An and Cynthia J. Burrows*

Department of Chemistry, University of Utah, 315 S. 1400 East, Rm 2020, Salt Lake City, Utah 84112-0850, USA. * Correspondence to: [email protected]

ABSTRACT

Oxidative damage to the G-rich telomeric sequence at the ends of chromosomes is proposed to be a significant contributor to cellular senescence. In this work, we address several questions related to oxidation of G quadruplexes in synthetic oligomers of the sequence d(TTAGGG)n.

INTRODUCTION

Estimates place the level of DNA oxidative damage to guanine in G-quadruplex sequences as a substantial fraction of the total guanine damage in the genome. Yet, the assessment of guanine oxidation as a function of genome location is complicated by the paucity of methods that provide both sequence information and structural identity of DNA lesions. We recently have taken advantage of a single-molecule technique in which ssDNA is electrophoretically driven through the �-hemolysin (�-HL) ion channel embedded in a lipid bilayer to analyze the presence of DNA lesions such as 8-oxo-7,8-dihydro-2’-deoxyguanosine (OG) and its further oxidation products (Sp, Gh) as well as for abasic sites (AP).1-3 This work examined the susceptibility of the 12 guanines of a telomere toward oxidation as a function of the fold (Na+ vs. K+), the nature of the oxidant, and the product outcome of the oxidation reactions. In addition, we synthetically incorporated lesions such as OG into specific locations of the telomere (Fig. 1) and monitored the effects on Tm values, CD spectra, and the translocation kinetics of individual strands through the �-HL nanopore.

RESULTS AND DISCUSSION In the present work, we find that the location of the G in the sequence and in the layers of the G-quadruplex influence the susceptibility of the G to oxidative damage. For example, one-electron oxidants such as CO3

.- show a strong preference for oxidation of the 5’G of a GGG sequence, and whereas G9 is a very reactive site when the telomere is

folded with K+, it less reactive in the presence of Na+ (for sequence, see Fig. 2). In contrast, 1O2 reacts nearly equally at all top and bottom faces of the stacked quadruplexes, but not in the

middle layer. Placement of OG in a top or bottom layer of the telomere results in destabilization and unfolding of that layer, whereas formation of OG in a middle layer leads to complete unfolding of the telomere (Fig. 2). These phenomena could be observed by changes in the Tm values and CD spectra. In addition, we monitored the translocation times of telomeric sequences through the a-HL ion channel and drew correlations between the rates of unfolding and the translocation times. These were highly dependent on the location of lesions in the sequence. CONCLUSIONS

The folded form of the telomere influences the reactivity of the various G sites as does the mechanism of oxidation (one-electron vs. singlet oxygen), and the G oxidation products in turn influence the resulting fold. Single-molecule experiments in which damaged DNA strands are threaded through the a-hemolysin nanopore help reveal the kinetics of unfolding of telomeres and may lead to single-molecule sequencing of damage sites.

REFERENCES 1. Schibel, A.E.P., An,

N., Jin, Q., Fleming, A.M., Burrows, C.J., White, H.S. J. Am. Chem. Soc. 2010, 132, 17992-17995.

2. 2. Schibel, A.E.P., Fleming, A.M., Jin, Q., An, N., Liu, J., Blakemore, C.P., White, H.S., Burrows, C.J., J. Am. Chem. Soc. 2011, 133, 14778–14784.

3. 3. An, N., Fleming, A.M., White, H.S., Burrows, C.J. Proc. Natl. Acad. Sci.(U.S.A.), revision submitted.

�

Figure 1. Oxidation of G destabilizes the quadruplex.

Figure 3. Electrophoretic translocation of DNA through �-


HOW DOES TELOMERASE RECOGNIZE ITS SUBSTRATES?

Sergei Gryaznov,1* Ronald Pruzan,1 Beata Kocon-Rebowska2 and Barbara Nawrot2 1 Geron Corp., 230 Constitution Drive, Menlo Park, CA 94025, USA, and 2 Centre of Molecular and Macromolecular

Studies, PAS, Lodz, Poland. * Correspondence to: [email protected]

ABSTRACT

Human telomerase demonstrates significantly higher affinity to telomeric d-(TTAGGG)2,3 substrates containing Rp-phosphorothioate internucleoside linkages than to their Sp-counterparts. Both Rp- and Sp-isomers of alpha-thio-dGTP were recognized by telomerase and incorporated into de novo synthesized DNA product, albeit less efficiently than dGTP, with the Rp-isomer showing markedly increased turnover rates over the Sp-isomer.

Telomerase is a unique reverse transcriptase responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cells. The enzyme is required for cell proliferative immortality however, and its activity has been detected in the vast majority (~85%) of human tumours. Hence, telomerase represents an attractive target for specific cancer cell-addressed therapy.

To learn more about the biochemistry of the enzyme and its interactions with substrates, we have examined the recognition of single-stranded oligodeoxynucleotides that serve as telomerase primers/substrates in vitro and in vivo. We have used highly purified telomerase and a two-primer competition assay for determining the dissociation rates of these primers. Primers/substrates having sequence permutations of d-(TTAGGG)n (n=2,3,4) were found to have considerably different telomerase affinities. These compounds formed complexes with telomerase having half-life values (t1/2) ranging from 14 min to greater than 1,200 min. A primer ending in the -GGG-3’ trinucleotide motif formed the most stable complex with the enzyme. We have found that interactions of telomerase with these native DNA substrates were mainly stabilized by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primers and the template region of telomerase RNA (hTR) apparently contributes less to the complex stability. The replacement of the native internucleoside phosphodiester (PO) by phosphorothioate (PS), N3’�P5’-phosphoramidate (NP), or N3’�P5’-thio-phosphoramidate (NPS) linkages in these primers altered their affinity to telomerase. The PS-oligo substrates with all-Rp-isomers demonstrated much higher complex stability than that for the all-Sp-counterparts, and the NPS-compounds have shown the highest affinity to telomerase. At the same time, both mix-Rp/Sp PS- and particularly mix-Rp/Sp NPS- compounds exhibit almost irreversible binding to telomerase, (or possibly a concomitant inhibition of the enzyme activity), as was judged by a non-exponential shape of telomerase-

substrate complex decay curves (Figure 1). In contrast, the NP-substrate did not form stable complex with telomerase.

Figure 1. Telomerase-oligonucleotide substrate dissociation curves. All the substrates are isosequential d(T2AG3)3 octadecamers.

Analogously to the natural dGTP, telomerase recognized and incorporated into de novo synthesized telomeric DNA products both Rp- and Sp-isomers of alpha-thio-dGTP, albeit less efficiently than it does with dGTP (Figure 2). The Rp-isomer showed markedly increased turnover rates over its Sp- counterpart, as reflected by the accumulation of d(TTAGGG)-TT*ApsG product (*A= 33P-lable). This is due to the formation of the PS- oligo product with Sp- internucleoside phosphorothioate group from the Rp- alpha-thio-dGTP (via an inversion of P-configuration during polymerization), following by its rapid dissociation from the enzyme.

Figure 2. PAGE analysis of characteristic telomeric DNA products obtained during telomerase extension of d(T2AG3)3 primer in the presence of either dGTP or alpha-thio-dGTP’s, and alpha-33P-dATP and TTP. The lowest major band corresponds to d(T2AG3)3-TTAG and all other bands to d(T2AG3)3-(T2AG3)n-TTAG products, respectively.

The results obtained provide further scientific rational for the design of potential oligonucleotide-based telomerase inhibitors with increased affinity to the enzyme.

Lanes: 1 - Rp- �S-dGTP 2 - Sp- �S-dGTP 3 - mix-Rp/Sp- �S-dGTP 4 - dGTP


Effects of fluorine and other substitutions on duplex and quadruplex structures studied by NMR

Nerea Martín-Pintado1, Maryam Yahyaee-Anzahaee2, Glen Deleavey2, Anna Aviñó3,

Modesto Orozco4, Ramón Eritja3, Masad J. Damha2, and Carlos González1*

1Instituto de Química Física Rocasolano, CSIC, C/ Serrano, 119, 28006 Madrid, Spain 2Department of Chemistry, McGill University, Montreal, QC, H3A 0B8, Canada

3Institute for Research in Biomedicine, IQAC-CSIC, 08028 Barcelona, Spain 4Joint IRB-BSC program on Computational Biology. Institute for Research in Biomedicine, and University of Barcelona.

08028 Barcelona, Spain E-mail: [email protected]

ABSTRACT

In this communication we will present our more recent results on structural studies of modified nucleic acids. We will focus on the effect of incorporating 2'-fluorine-modified nucleotides and 8-amino guanines in duplex and quadruplex structures.

Advances in synthetic chemistry afford new nucleic acids with intriguing properties. In this communication, we will present some of our studies on sugar-modified nucleic acids, and 8-amino substitution in guanines. In particular, nucleic acids analogs containing 2'-fluoro-arabino (2'F-ANA) and 2'-fluoro-ribose (2'F-RNA) are interesting compounds for their potential applications in antisense and interference RNA therapy. In addition, arabino nucleic acids (ANA) have become attractive systems to construct genetic systems based on alternative chemical platforms. The preferential conformations of these analogs are diferent: ANA and 2'F-ANA are considered to be DNA analogs, while 2'F-RNA is considered as an RNA-like nucleotide. By changing the pattern of incorporation of these analogs in a particular oligonucleotide sequence, structure and stability, as well as binding affinity for RNA targets can be tuned. In this communication, we discuss the three-dimensional structure of several chimeric and hybrid duplexes, whose sequences combine different patterns of ANA, 2'F-ANA and 2'F-RNA nucleotides, as determined by combining 1H and 19F NMR spectroscopy. The effect of fluorine substitutions on sugars is not only limited to double stranded structures. Their effect on the structure and stability of guanine quadruplexes is also very interesting. 2'F-ANA substitutions can be stabilizing or destabilizing, depending on the particular topology of the quadruplex. In human telomeric sequences this effect is dramatic. Whereas the native sequence can adopt different conformations depending on the experimental conditions, a single 2'F-ANA substitution provokes the formation of a very stable parallel propeller structure. We discuss here the solution structure of a 2'F-ANA substituted quadruplex. Finally, we will also present our most recent results on 8-

amino-substituted guanine quadruplexes. Base-modifications in G-tetrads are usually not well-tolerated and have a destabilization effect in G-quadruplexes. One of the few exceptions is the substitution by 8-amino-guanines. 8-amino-Gs stabilize triplexes and parallel-hairpins. However, its effect on the stability on G-quadruplexes is not clear, and depends on the quadruplex topology and the sequence context. In the case of tetramolecular parallel quadruplexes, such as that formed by d(TGGGGT), we have found that substitution of the first guanine by a 8-amino-guanine provokes the formation of an unsual quadruplex dimer, stabilized G:T:G:T and g:T:g:T tetrads. Dimerization of the two quadruplexes occurs in an antiparallel orientation through their 5'-side as shown in the figure below.

Figure 1. A) Propeller quadruplex stabilized by 2'F-ANA

nucleotides. B) Parallel quadruplex dimer stabilized by 8-amino guanines.

A B 3

5

5

3

Tuesday, 7th August 1.40 pm

CLICK NUCLEIC ACID LIGATION: CHEMISTRY, BIOCHEMISTRY AND APPLICATIONS Afaf El-Sagheer1,2, Montserrat Shelbourne1, Xiong Chen1 and Tom Brown1

1School of Chemistry, University of Southampton SO17 1BJ. UK. 2 Chemistry Branch, Dept. of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez, 43721, Egypt.

Correspondence to: [email protected]

ABSTRACT

Click ligation utilizes the copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction). It is an efficient method of joining together DNA and RNA strands and has been used for the synthesis of cyclic oligonucleotides,(1-3) oligonucleotide catenanes,(2) very stable cyclic mini-duplexes,(1) duplexes that are linked across the major groove,(4) covalently fixed DNA nanoconstructs(5) and large RNA constructs.(6) The method produces an unnatural DNA backbone linkage that can be varied by changing the structures of the participating alkyne and azide.(7) Careful design produced a biocompatible DNA backbone (Figure 1) that can be read through by DNA(8) and RNA polymerases.(9) A high-resolution NMR study revealed that the linkage in Figure 1B is accommodated in a B-DNA helix with minor distortion.(10)

Figure 1. First generation triazole DNA (A), second generation biocompatible linkage (B) and normal DNA (C).

Figure 2. a. The ring strain promoted alkyne-azide cycloaddition reaction (SPAAC reaction) for click DNA ligation between azide and cyclooctyne-labeled oligonucleotides. b. Chemical structure of DIBO triazole at the ligation point.

Copper-free click DNA strand ligation and crosslinking can also be carried out if strained cyclooctyne analogues are used (Figure 2).(11) This method has the advantage of being potentially valuable for in vivo applications as it does not require metal ion catalysis.

REFERENCES 1. El-Sagheer, A.H., Kumar, R., Findlow, S., Werner, J.M.,

Lane, A.N. and Brown, T. (2008) A very stable cyclic DNA miniduplex with just two base pairs. Chembiochem, 9, 50-52.

2. Kumar, R., El-Sagheer, A.H., Tumpane, J., Lincoln, P., Wilhelmsson, L.M. and Brown, T. (2007) Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry. J. Am. Chem. Soc., 129, 6859-6864.

3. El-Sagheer, A.H. and Brown, T. (2008) Synthesis, Serum Stability and Cell Uptake of Cyclic and Hairpin Decoy Oligonucleotides for TCF/LEF and GLI Transcription Factors. Int. J. Peptide Res.Therapeut., 14, 367-372.

4. Kocalka, P., El-Sagheer, A.H. and Brown, T. (2008) Rapid and efficient DNA strand cross-linking by click chemistry. Chembiochem, 9, 1280-1285.

5. Lundberg, E.P., El-Sagheer, A.H., Kocalka, P., Wilhelmsson, L.M., Brown, T. and Norden, B. (2010) A new fixation strategy for addressable nano-network building blocks. Chem. Commun., 46, 3714-3716.

6. El-Sagheer, A.H. and Brown, T. (2010) New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes. Proc. Natl. Acad. Sci. U. S. A., 107, 15329-15334.

7. El-Sagheer, A.H. and Brown, T. (2009) Synthesis and Polymerase Chain Reaction Amplification of DNA Strands Containing an Unnatural Triazole Linkage. J. Am. Chem. Soc., 131, 3958-3964.

8. El-Sagheer, A.H., Sanzone, A.P., Gao, R., Tavassoli, A. and Brown, T. (2011) Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in E. coli. Proc. Natl. Acad. Sci. U. S. A., 108, 11338–11343.

9. El-Sagheer, A.H. and Brown, T. (2011) Efficient RNA synthesis by in vitro transcription of a triazole-modified DNA template. Chem. Commun., 47, 12057-12058.

10. Dallmann, A., El-Sagheer, A.H., Dehmel, L., Mügge, C., Griesinger, C., Ernsting, N.P. and Brown, T. (2011) Structure and dynamics of triazole-linked DNA - biocompatibility explained. Chemistry - A European Journal, 17, 14714-14717.

11. Shelbourne, M., Chen, X., Brown, T. and El-Sagheer, A.H. (2011) Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction. Chem. Commun., 47, 6257-6259.


NEW DNA ANALOGS – SYNTHESIS, BIOLOGICAL, AND CHEMICAL PROPERTIES

Marvin H. Caruthers1* 1University of Colorado, Department of Chemistry & Biochemistry, Boulder, CO 80309 * Correspondence to:

[email protected]

ABSTRACT

The synthesis of DNAs having triazoylphosphonate[1], boranephosphamidate[2], boranealkylphosphine[3] and boranephosphonate[4] internucleotide linkages will be described. These analogs have unique biological properties while those having borane reduce metals.

INTRODUCTION

Chemically modified, synthetic oligonucleotides (ODNs) act in a sequence-specific manner to modulate gene expression through their use as antisense reagents, triple-helix-forming oligomers, siRNAs, antagomers, ribozymes, and CpG immunostimulatory reagents1. ODNs have also been used for diagnostic and nanomaterial applications2. Here we describe new analogs that can be readily synthesized and have unique biological and chemical properties.

RESULTS AND DISCUSSION

Copper (I) catalyzed azide-alkyne [3+2] cycloaddition chemistry (CLICK) was used to join triazole to a phosphonate internucleotide linkage and generate triazoylphosphonate (1,R1=H) ODNs. The synthons were protected 2’-deoxynucleoside 3’-O-(N,N-diisopropylamino)ethynylphosphines. When used with the standard solid-phase DNA synthesis cycle, appropriately protected 2’-deoxynucleoside 3’-O-phosphoramidites, and 5-ethylthio-1H-tetrazole, ODNs having ethynylphosphonate internucleotide linkages at predetermined sites were generated. Post synthesis, triazole was introduced from the appropriate azide and CLICK chemistry. Of particular interest was the observation that a fluorescently labelled 16 mer carrying as few as four triazoylphosphonate internucleotide linkages had unique sub-cellular distribution patterns when transfected without lipid into different cell lines. In HeLa cells the ODN was localized in the nucleus whereas with WM-239 cells (metastatic melanoma) localization was highly diffused in the cytoplasm. In Jurkat cells (T-lymphocyte), fluorescence was cytoplasmic but punctate and diffuse. In a neuroblastoma cell line (SK-N-F1), fluorescence was diffuse and cytoplasmic. These triazoylphosphonate ODNs were active as microRNA antagomers..

Boranephosphamidate ODNs[2] were prepared as dimers and then incorporated into DNA. Synthesis begins with 3’-O-bis(dialkylamino)-2’-deoxynucleoside. Condensation generates the appropriate PIII dimer which is boranated,

isolated, and converted to a dimer 3’-O-phosphoramidite. This dimer is used to prepare ODNs. These analogs are nuclease resistant and form stable duplexes with DNA.

The synthesis of boranemethylphosphine DNA[3] has recently been reported3. These ODNs are nuclease resistant, form duplexes with RNA and can be transfected without lipid into HeLa cells. Under physiological salt conditions, duplexes having these ODNs do not have suppressed Tms.

Boranephosphonate DNA has been synthesized using a new, unpublished procedure. The approach uses silyl protection on the nucleobases and a 5’-dimethoxytrityl as the transient protecting group. Unlike previous procedures, this method allows synthesis with

standard phosphoramidites on supports, including acidic removal of trityl groups.

Borane containing ODNs reduce several metal ions. For example 4 reduced Au3+ and PtCl4

- at room temperature, whereas Ag+ was reduced at 55�C over several hours. The products of these reductions were the metals, boronic acid, and intact, natural internucleotide linkages. Recently this type of chemistry has been used to prepare phosphate esters from borane phosphonate DNA. Thus we anticipate that borane phosphonates can be used as phosphate protecting groups for the preparation of analogs unavailable by other chemistries. CONCLUSION

We report the first synthesis of three analogs (1, 2, 3) and a new method for preparing 4. These ODNs exhibit several unique biological and chemical properties. DNA metal reduction could prove useful for designing nanomaterials and for introducing new DNA analogs at phosphorus.

REFERENCES 1. McDermott, A. M., Heneghan, H. M., Miller, N., and

Kerin, M. J. Pharm. Res. 2011,28, 3016-3029. 2. Seeman, N. C. Annu. Rev. Biochem. 2010,79, 65-87. 3. Krishna, H. and Caruthers, M. H. J. Am. Chem. Soc.

2011,133, 9844-9854.


SYNTHESIS OF CONFORMATIONALLY CONSTRAINED OLIGONUCLEOTIDE STRUCTURES

Enrique Pedroso,* Roger Tresánchez, Albert Sánchez, Carlos Rodergas, Roser Borràs and Anna Grandas

Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, Barcelona, Spain. *Correspondence to: [email protected]

ABSTRACT Two novel methods for the easy incorporation of

conformational constraints into synthetic oligonucleotides have been devised: i) cyclization between a maleimide and a thiol placed at the ends of the oligonucleotide chain, ii) interstrand cross-linking between two nucleobases.

Our first appearing picture of nucleic acids structure

visualizes DNA as the Watson-Crick double-stranded helix, whereas RNA is depicted as an ensemble of single-stranded segments together with double-stranded regions, hairpins, internal and terminal loops, bulges, etc. However, DNA has also the potential to fold into a variety of non-B DNA conformations such as Z-DNA, hairpins, triplexes, G-quadruplexes, i-motifs, cruciforms and A-motifs.1

The common feature of most non-B DNA conformations is that they are constituted by repetitive DNA sequences. These alternative conformations affect key genetic events, such as DNA replication, transcription, recombination and repair, inducing genome instability and eventually leading to human diseases.2 For instance, in the neurological diseases caused by triple repeat expansions,3 expanded DNA repeats form unusual non-B DNA structures and, in some cases, long mismatched hairpins of the RNA transcripts are involved in the pathogenesis of the disorders.4,5

In this context, our understanding of the conformations of large DNAs and RNAs using short oligonucleotide models is hampered by the low structural and thermal stability of the latter compared to the native nucleic acids. Therefore, the incorporation of conformational constraints into the oligonucleotide constructs may facilitate their structural study. In this communication we will report on two different chemical tools that restrict the conformational space accessible to synthetic oligonucleotides.

Cyclization has been recognized as a way to afford oligonucleotide constructs that are especially useful in thermodynamic studies because they denaturate in a monomolecular fashion. Yet, the synthesis of circular DNAs formed by repetitive sequences has proved to be a challenging task.6

Now, taking advantage of our recently described solid-phase method to attach maleimides to the 5' end of an oligonucleotide chain,7 we have devised a novel cyclization method. This is achieved by incorporating a protected maleimide at the 5' end and a protected thiol at

the 3' end of a linear precursor using commercially available reagents. Careful deprotection of the maleimide and the thiol promotes intramolecular cyclization cleanly and in high yield through a Michael-type addition. The method does not rely on internal or template-assisted preorganization of the precursor oligonucleotide, so it is applicable to any sequence provided that high dilution reaction conditions are used.

Figure 1. General structure of the cyclic oligonucleotides. Another well-established approach to modify nucleic

acid structure in order to reduce its conformational mobility is to introduce a cross-link. Furthermore, cross-links are formed as a result of therapeutic or environmental agents, and synthetic cross-linked oligonucleotides can be used to study their effects on nucleic acid structure as well as to get insight into the repair mechanisms.8

We will describe our preliminary results in developing a novel and easy method to generate interstrand nucleic acid cross-links. The cross-link does not significantly perturb B-DNA conformation and has one of the desired requirements of ideal cross-links, which is to be reversible.

REFERENCES

1. Choi, J., Majima, T., Chem. Soc. Rev. 2011, 40, 5893-5909.

2. Bacolla, A., Wells, R.D., J. Biol. Chem. 2004, 279, 47411-47414.

3. Mirkin, S.M., Nature 2007, 447, 932-940. 4. Orr, H.T., Zoghbi, H.Y., Annu. Rev. Neurosci. 2007,

30, 575-621. 5. Krzyzosiak, W.J., Sobczak, K., Wojciechowska, M.,

Fiszer, A., Mykowska, A., Kozlowski, P., Nucleic Acids Res. 2012, 40, 11-26.

6. Hartig, J.S., Kool, E.T., Nucleic Acids Res. 2004, 32, e152.

7. Sánchez, A., Pedroso, E., Grandas, A., Org. Lett., 2011, 13, 4364-4367.

8. Noll, D.M., Noronha, A.M., Wilds, C.J., Miller, P.S., Front. Biosci. 2004, 9, 421-437.


NUCLEIC ACID STRUCTURES BASED ON DOUBLE-HEADED NUCLEOSIDES

Poul Nielsen,*,1 Pawan Kumar,1 Charlotte S. Madsen,1 Pawan K. Sharma,2 Sarah Witzke,1 and Michael Petersen1

1 Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark. 2 Department of Chemistry, Kurukshetra University, Kurukshetra-136 119, India.

* Correspondence to: [email protected]

ABSTRACT

Double-headed nucleosides are nucleosides with additional nucleobases. The recognition potential of these can be used in the design of artificial nucleic acid motifs. When introduced in a DNA duplex, a double-headed nucleoside with an additional nucleobase in the 2´-C-position can form two Watson-Crick base pairs with complementary bases in the duplex core.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

In recent years, we and others have prepared a series of double-headed nucleosides with the purpose of using the recognition potential of the additional nucleobases in various nucleic acid constructs [1-5]. For instance, a thymidine nucleoside with an additional thymine in the 5´-position, demonstrated a base-base interaction in the minor groove of DNA, when incorporated specifically in the two complementary strands in a so-called (–3)-zipper orientation [3,4]. Herein we present the double-headed nucleosides 1 and 2 (Fig. 1) with the second nucleobase in the 2´-position and hereby the potential to enter into the interior of the duplex.

When introduced into oligonucleotides and studied in different duplexes with UV melting experiments, the double-headed nucleoside 1 was found to work as a compressed dinucleotide mimic with both nucleobases forming Watson-Crick base pairs with complementary adenines (Fig. 2(a)) [5]. Mis-matched nucleosides in the complementary strand were discriminated, and the motif could be repeated in the duplex. Furthermore, surprisingly stable duplexes were obtained when monomer 1 was introduced in each of the complementary strands in either a (–2), (–1) or (+1)-zipper orientation. In the latter, a T-T base pair is formed centrally in the duplex (Fig. 2(b)) and the thermal

stability is significantly increased as compared to a duplex with an ordinary T-T mismatch. Also this motif can be repeated in the duplex. In all cases the additional thymines of 1 intercalates in the duplex in the 3´-postion to the uracil. MD-simulations and NMR-spectroscopy supported the observations from the melting experiments

(Fig. 2). The synthesis of 1 has been accomplished from a 9-

step procedure starting with D-ribose [5]. However, we envisioned that the presence of a 2´-hydroxygroup might not influence the ability of the two nucleobases to base pair in the duplex. Hence the 2´-arabino nucleoside 2 was prepared in a convenient 5-step procedure from uridine. In the melting experiments, the duplexes containing 2 demonstrated the same structural properties as those with 1.

The new nucleic acid motifs obtained with 1 and 2 (or analogues with other nuclebase combinations) extend the DNA recognition repertoire and might constitute a new framework for transferring molecular information.

REFERENCES 1. Pedersen, S. L., Nielsen, P. Org. Biomol. Chem.

2005, 3, 3570-3575. 2. Wu, T., Nauwelaerts, K., Van Aershot, A., Froeyen,

M., Lescrinier, E., Herdewijn, P. J. Org. Chem. 2006, 71, 5423-5431.

3. Christensen, M. S., Madsen, C. M., Nielsen, P. Org. Biomol. Chem. 2007, 5, 1586-1594.

4. Shaikh, K. I., Madsen, C. S., Nielsen, L. J., Jørgensen, A. S., Nielsen, H., Petersen, M., Nielsen, P. Chem. Eur. J. 2010, 16, 12904-12919.

5. Madsen, C.S., Witzke, S., Kumar, P., Negi, K., Sharma, P.K., Petersen, M., Nielsen, P. Chem. Eur. J. in press

Figure 1. Two double-headed nucleosides.

Figure 2. Representative snapshots from MD-simulations of two different nucleic acid motifs formed by 1. (a) the 5�-UT:3�-AA motif and (b) the 5�-UTA:3�-AUT motif.

(a) (b)


FURAN-BASED OLIGONUCLEOTIDES FOR CROSS-LINKING AT THE DNA/DNA AND DNA/PROTEIN INTERFACE

Carrette, L.L.G., Gyssels, E., Op de Beeck, M., and Madder, A. Laboratory for Organic and Biomimetic Chemistry, UGent, Krijgslaan 281, S4, 9000, Gent, Belgium

ABSTRACT

Our group has developed a cross-link strategy based on the incorporation of a furan moiety as a caged reactive entity into oligodeoxynucleotides (ODNs). These furan-modified nucleic acids form cross-links upon selective furan oxidation. We here describe a series of applications of the developed probes, from nucleic acid duplex and triplex cross-linking to DNA-protein cross-linking.

INTRODUCTION

Recently a novel cross-linking strategy was developed, inspired by the toxic liver metabolism of furan. Furan and analogues are oxidised in the liver by Cyt P450 enzymes to

butene-dial or 4-oxo-butenals.

These very reactive

intermediates quickly react with proximate

nucleophiles from proteins and DNA eliciting a toxic response.

We have shown that furan-oxidation based cross-linking occurs in a fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated.

RESULTS AND DISCUSSION

Several furan modified nucleosides have been synthesized for incorporation in reactive nucleic acids, allowing for a variable positioning of the furan unit in the duplex [1-4].

A detailed investigation of sequence selectivity and linker influence has been carried out and thorough structural characterization has shed light on the exact crosslinking mechanism. Whereas building blocks 1 through 4 consistently show cross-linking to complementary A or C bases, building block 5 reveals an unprecedented C-selectivity.

The position and linker by which the furan moiety is attached were shown to significantly influence the crosslinking properties of the furan-modified ODN towards complementary sequences (interstrand duplex crosslinking), host duplexes (interstrand triplex crosslinking) or proteins (DNA/protein crosslinking). Using building block 6 proof of principle for selective DNA-protein cross-linking was obtained.

Figure 3. Furan mediated peptide-DNA crosslinking

CONCLUSION

A combination of features renders the furan-oxidation crosslink methodology very attractive. Benefitting from an inducible reactivity principle, only equimolar amounts of modified strands are necessary to produce substantial amounts of crosslinked material without collateral damage. Formation of stable crosslinked products is fast and purification is facile in view of the ‘self-destructing’ behaviour of the furan-modified strands. Fine-tuning of duplex or triplex stability, crosslink selectivity and yield is possible depending on the specific choice of building block and desired target. Furthermore, a biocompatible oxidative triggering signal having been developed, the way to biological applications is currently open.

REFERENCES 1. Op de Beeck, M., Madder, A., J. Amer. Chem. Soc., 2011, 133, 796-807. 2. Stevens, K., Madder, A. et al. Chem. Eur. J., 2011, 6940-6953. 3. Jawalekar, A.M., Madder, A. et al. Chem. Comm., 2011, 2796-2798. 4. Stevens, K., Madder, A., Nucleic Acids Res., 2009, 1555-

1565.

5'

5'

3'

3'

5'

5'

3'

3'

5'

5'

3'

3'

oxidationcrosslinkformation

O O O

Figure 1. Furan-oxidation triggered nucleic acid cross-linking

NH

O

ONO

OH

HO

NH

OO

1

HO

HO

O

2

HO

HO

O

O

3

OHO

OH

O

4

NH

O

ONO

OH

HO

NHHN

OO

5

NH

O

ONO

OH

HO

6

O

Figure 2. Furan-modified nucleoside

analogues

O

OH

HO

N

NH

O

O

O

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