See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7928847
Structural features of a central mismatch in oligonucleotide hybrid duplexes visualized via Raman spectroscopy... Article in Biopolymers · October 2005 DOI: 10.1002/bip.20277 · Source: PubMed
CITATIONS
READS
3
20
4 authors: Daniel Nemecek
Hana Vaisocherova
Central European Institute of Technology-Cz…
The Czech Academy of Sciences
28 PUBLICATIONS 222 CITATIONS
36 PUBLICATIONS 1,452 CITATIONS
SEE PROFILE
SEE PROFILE
Josef Štěpánek
Pierre-Yves Turpin
Charles University in Prague
Pierre and Marie Curie University - Paris 6
108 PUBLICATIONS 889 CITATIONS
186 PUBLICATIONS 2,195 CITATIONS
SEE PROFILE
All content following this page was uploaded by Daniel Nemecek on 10 July 2015.
The user has requested enhancement of the downloaded file.
SEE PROFILE
Daniel Ne˘mec˘ek1,2* Hana Vaisocherova´2 Josef S˘te˘pa´nek2 1
Pierre-Yves Turpin1
Universite´ P. et M. Curie, L.P.B.C. (CNRS UMR 7033), 4 place Jussieu, Case 138, F–752 52 Paris Cedex 05, France 2
Institute of Physics, Charles University, Ke Karlovu 5, Praha 2, CZ-121 16, Czech Republic
Structural Features of a Central Mismatch in Oligonucleotide Hybrid Duplexes Visualized Via Raman Spectroscopy: Model System for Evaluation of Potential ‘‘Antisense’’ Drugs
Received 25 August 2004; revised 15 February 2005; accepted 16 February 2005 Published online 1 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20277
Abstract: Structural features of mismatched base pairs were studied on four nonamer hybrid duplexes formed between the 50 -d(GTGATATGC)-30 complement and its 50 -r(GCAUNUCAC)-30 (N ¼ A, C, G, U) counterparts. This oligonucleotide set is considered a model molecular system for future systematic studies of various modifications of internucleotide linkages with respect to their impact on the structure of mismatched base pairs. Raman spectra, measured at 158C, revealed the prevailing A-like structure of the RNA strand and mixed A-like and B-like characteristics for the DNA strand. All three mismatches disturb only weakly the overall conformation of the hybrid duplex in contrast to analogous mismatched DNA duplexes. In particular, the dT rG mismatch influences the global hybrid duplex geometry almost negligibly. The dT rC and dT rU mismatches induce somewhat more pronounced distortions of the backbone structure and of the thymine position, the latter being expressed by a change of the surrounding methylene group without effect on the carbonyl’s vibrations. Structural effects of the mismatches correlate well with the duplex thermodynamic stabilities obtained by ultraviolet (UV) absorption, i.e., the dT rG mismatch decreases the hybrid duplex stability very weakly while the effect of both pyrimidine–pyrimidine mismatches is considerable. # 2005 Wiley Periodicals, Inc. Biopolymers 79: 1–8, 2005 This article was originally published online as an accepted preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at
[email protected] Keywords:
nucleic acid; oligonucleotide; Raman spectroscopy; mismatch; hybrid duplex; antisense
Correspondence to: Daniel Ne˘mec˘ek; e-mail: nemecekd@umkc. edu *Current address: UMKC School of Biological Sciences, Division of Cell Biology and Biophysics, 5100 Rockhill Road, Kansas City, MO 64110-2499 Contract grant sponsor: Grant Agency of Charles University (GACU) and Ministry of Education of the Czech Republic (MECR) Contract grant numbers: 310/2004 (GACU) and MSM 113200001 (MECR) Biopolymers, Vol. 79, 1–8 (2005) 2005 Wiley Periodicals, Inc.
#
1
2
Ne˘mec˘ek et al.
INTRODUCTION Several molecular biological techniques require accurate predictions of matched v. mismatched hybridization thermodynamics (e.g., polymerase chain reaction (PCR), sequencing by hybridization, gene diagnostics, antisense strategy). Therefore, thermodynamics and molecular structures of Watson–Crick and mismatch base pairs have been extensively studied by methods of ultraviolet (UV) melting,1,2 nuclear magnetic resonance (NMR) distance geometry,3–6 and X-ray diffraction.7–9 Single rG rU or dG dU mismatches can form stable wobble base pairs10,11 that are almost as stable as rA : rU or dA : dT Watson–Crick base pairs.4 dG dT and dT dT mismatch base pairs likely form wobble base pairs as well, whereas dC dT mismatch is rather stabilized by stacking interactions with adjacent purine rings. Structural studies indicate that hydrogen bonds are formed between mismatched nucleotides and that water molecules can be involved in base pairing too. Stability of hybrid duplexes with a single mismatch is affected by the nature of the mismatch and the adjacent base pairs.1 It is also different for dN rN0 and dN0 rN mismatches. The rG dT mismatches are the most stable ones, whereas the stability of rU dT and rC dT mismatches was found to be weak.1 Study of mismatch structures is also important for the development of new synthetic drugs based on a precise recognition of target molecules (i.e., proteins or nucleic acids). They are nowadays intensively searched and investigated in order to improve drug efficiency and to suppress side effects. ‘‘Antisense’’ strategy is a concept of novel gene therapy in which the translation of a pathogen gene is inhibited by a short oligonucleotide complementary to the mRNA target sequence. It forms a local double-helical complex that prevents the mRNA to serve as a matrix for deleterious protein synthesis.12 If the ‘‘antisense’’ oligonucleotide is the deoxy type, the effectiveness of the antisense drug can be substantially enhanced by the RNase H enzyme that excises the mRNA strand of the hybrid duplex while the ‘‘antisense’’ deoxy strand is left unperturbed. Natural oligonucleotides are unfortunately unsuitable for in vivo applications owing to their lack of resistance against cell nucleases. An intense search is therefore underway to find novel synthetic oligonucleotide analogues that would possess suitable properties, i.e., resistance in a cellular environment, high binding affinity, specificity to the target nucleic acid sequence, and finally elicitation of RNase H activity. We have investigated thermodynamic properties of new types of internucleotide linkage modifications by Raman spectroscopy, in developing a method that would allow the determination of the stoichiometry,
stability, and structure of the complexes formed between a modified oligonucleotide chain and its natural complementary counterpart. This approach, based on precise Raman titration, Raman temperature profile measurements, and special data treatment procedures, has been applied to characterize two families of various isopolar, nonisosteric modifications of internucleotide linkages.13,14 Investigation of the binding specificity, i.e., whether the particular internucleotide linkage modification does or not favor ‘‘fake’’ base pairing, is a natural continuation of our studies. The first Raman results were obtained for a model system of DNA duplexes15 based on 50 -d(GTGATATGC)-30 and 50 -d(GCATNTCAC)-30 strands, the latter with alternated central nucleobase N ¼ A, C, G, T. We confirmed the sensitivity of Raman spectra to structural deviations of mismatch duplexes and we obtained reference spectral signatures on a complete set of natural deoxyoligonucleotides. As a next step, this work presents a study of mismatch hybrid duplexes based on the analogous model system of 50 -d(GTGATATGC)-30 and 50 -r(GCAUNUCAC)-30 strands, N ¼ A, C, G, U. The emphasis of the study was to determine structural changes in hybrid duplexes due to a mismatched base pair and to obtain their Raman spectral signatures. The structural characteristics were compared with the duplex stabilities determined by UV absorption measurements.
MATERIAL AND METHODS The deoxyoligonucleotide complement was synthesized in LMFR of Masaryk University in Brno, then high performance liquid chromatography (HPLC) purified and lyophilized. RNA oligonucleotides were purchased from Dharmacon, Inc., in the stable 20 -acetoxyethoxy protected form (for oligonucleotide sequences, see Table I). They were deprotected with enclosed buffer and lyophilized. Stock solutions of the single-stranded oligonucleotides (40 L) were prepared by dissolution in a Millipore-filtered solution of 4 mM Na2SO4 and 100 mM NaCl. The concentration was 20 mM in nucleotides (UV determination). DNA–RNA mixed solutions of 1:1 molar ratio were prepared from the single-strand solutions by weighting. After Raman measTable I Composition of the Studied Oligonucleotides Oligonucleotide DNA complement RNA1 RNA2 RNA3 RNA4
Sequence 50 -d(GTG ATA TGC)-30 50 -r(GCA UAU CAC)-30 50 -r(GCA UCU CAC)-30 50 -r(GCA UGU CAC)-30 50 -r(GCA UUU CAC)-30
Mismatch in Oligonucleotide Hybrid Duplexes
3
Table II Thermodynamic Parameters of Nonamer Hybrid Duplexes with Matched or Mismatched Central Base Pair Obtained from UV Absorptiona
Duplex (Central bp) dT rAb dT rCc dT rGb dT rUc
DS0 (cal-mol1 K1)
DH0 (kcal mol1)
210 6 10 193 6 19 187 6 9 187 6 16
72 6 3 62 6 6 64 6 3 61 6 5
Tm (8C)
0 DG310 k (kcal mol1)
Ctot ¼ 80 M
7.08 6 0.06 2.68 6 0.28 6.15 6 0.05 3.50 6 0.17
38.7 6 0.2 18.3 6 0.6 34.4 6 0.2 21.6 6 0.6
Ctot ¼ 4 M 30.9 6 0.2