CA2218443A1 - Metal-containing dna - Google Patents

Metal-containing dna Download PDF

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CA2218443A1
CA2218443A1 CA002218443A CA2218443A CA2218443A1 CA 2218443 A1 CA2218443 A1 CA 2218443A1 CA 002218443 A CA002218443 A CA 002218443A CA 2218443 A CA2218443 A CA 2218443A CA 2218443 A1 CA2218443 A1 CA 2218443A1
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dna
metal
duplex
containing dna
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Palok Aich
Jeremy S. Lee
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University of Saskatchewan
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Priority to CA002218443A priority Critical patent/CA2218443A1/en
Priority to CA002229386A priority patent/CA2229386A1/en
Priority to CA002255113A priority patent/CA2255113C/en
Priority to ES98960961T priority patent/ES2210847T3/en
Priority to EP98960961A priority patent/EP1042344B1/en
Priority to PCT/CA1998/001149 priority patent/WO1999031115A1/en
Priority to DE69819780T priority patent/DE69819780T2/en
Priority to AT98960961T priority patent/ATE254134T1/en
Priority to AU16562/99A priority patent/AU1656299A/en
Priority to US09/213,052 priority patent/US6432641B1/en
Priority to JP2000539038A priority patent/JP2002508386A/en
Priority to EP03023704A priority patent/EP1382610A3/en
Publication of CA2218443A1 publication Critical patent/CA2218443A1/en
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    • G11C13/0009—RRAM elements whose operation depends upon chemical change
    • G11C13/0014—RRAM elements whose operation depends upon chemical change comprising cells based on organic memory material
    • G11C13/0019—RRAM elements whose operation depends upon chemical change comprising cells based on organic memory material comprising bio-molecules
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    • G11C13/00—Digital stores characterised by the use of storage elements not covered by groups G11C11/00, G11C23/00, or G11C25/00
    • G11C13/0002—Digital stores characterised by the use of storage elements not covered by groups G11C11/00, G11C23/00, or G11C25/00 using resistive RAM [RRAM] elements
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Abstract

The invention provides a DNA molecule comprising a metal-containing DNA molecule, an electron donor electrically coupled to a first end of the metal-containing DNA duplex and an electron-acceptor electrically coupled to a second end of the metal-containing DNA duplex, the metal-containing DNA duplex comprising a divalent metal cation substituted for the imino protons of a DNA base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+, Co2+, and Ni2+.

Description

M-DNA
Jererny S. ~.ee Biochemistry. U of S.
As described in the attached paper, M-DNA behaves as a molecular wire. This property makes it ideal for the design of microelectronic circuits for the nanotechnology of the future.

In addition, M-DNAis nuclease resistant and therefore it will not be subject to rapid destruction as is the case with unmodified DNA.
As well, M-DNAis less negatively-charged than normal DNA and therefore it will be able to penetrate the cell membrane more easily. Once inside the cellit will slowly convert back to normal DNA. These attributes would be useful in the following technologies:
[a] DNA immunization. Plasmid DNA can be injected into the muscle of animals either directly or with a gene gun. In the muscle cells, the genes within the plasmid are expressed to produce proteins to which the animal mounts an antibody response. Thus the vaccine is produced by the animal itself. Because M-DNAis nuclease resistant it would be expected to produce a more sustained immune response.
[b] Antisense or antigene technology. Short oligonucleotides which are complimentary to a specific gene sequence have been shown to inhibit the production of that gene product. However, the oligonucleotide must be able to enter the cell and survive attack from ubiquitous n-l~leases. Therefore, conversion of the oligonucleotide to M-DNA would serve as a very useful but general delivery system.

It has been proposed that the stacked aromatic bases of DNA may act as a '~
way' for the efficient transfer of electrons 1,2,3, For example, it has been demonstrated that photoinduced electron transfer occurred between two metallointercalators tethered at either end of a 15-base pair duplex 4. On the other hand, kinetic analysis of distance-dependent elect~on transfer in a DNA hairpin sugges~ed that DNA is only somewhat more effective than proteins as a conductor of electrons 5~6. We report here that M-DNA not only contains the ~ stack but also an interchelated metal ion (Zn2+, Co2+, or Ni2+) 7, nlal~in~ it an excellent candidate for the conductance of electrons. Efficient electron transfer is observed between two fluorophores separated by 54 base pairs (over 150 A) in an M-DNA duplex.
Moreover, addition of a sequence-specific DNA-binding protein prevents the flow of electrons. Therefore, M-DNA behaves as a molecular wire and could be manipulated to prepare self-assembling electronic circuits.

M-DNA is formed at pHs above 8 in the presence of Zn2+, M2+, and Co2+ but not Mg2+ or Ca2+ 7. All bacterial and synthetic DNA (except perhaps poly[d(AT)])dismutate to M-DNA under these conditions but the process is readily reversible by lowering the pH and/or addition of EDTA. Unlike B-DNA, ethidium will not bind to M-DNA and this forms the basis of a rapid and sensitive "ethidium fluorescence assay" to monitor M-DNA formation. The mobility of linear or covalently closed circular forms of M-DNA in agarose gels was only slightly less than that of B-DNA, ruling out the possibility that the metal ions were causing condensation or aggregation of the DNA. NMR studies showed that the imino protons of T (pKa 9.9)and G (pKa 9.4) were not present in M-DNA explaining the requirement for a high pH and suggesting that they were replaced by the metal ion7. Alternatively, the imino protons might be opaque to NMR due to rapid exchange with solvent. To distinguish between these possibilities the release of protons was monitored during the formation of M-DNA. As shown in Figure 1, M-DNA begins to form at about 0.7 mM NiCl2 (as judged from the ethidium fluorescence assay); there is a concomitant release of protons so that KOH must be added to maintain the pH at 8.5. At 1.8 mM
NiCl2, M-DNA formation is virtually complete and the complex starts to precipitate.
Therefore, one proton is released per Ni2+ atom per base pair during the formation of M-DNA. The Zn2+ and Co2+ isomers of M-DNA also release protons during formation but precipitation of the complex occurs at a lower concentration of divalent metal ion than with Ni2+. These results are only consistent with the metal ion being coordinated to the N3 position of T and N1 of G in every base pair.
Based on these observations, a proposed structure for M-DNA can be modelled as shown in Figure 2. The A-T and GC base pairs are isomorphous which is a common feature of all stable helical nucleic acid structures8 9. Compared to a Watson-Crick base pair, insertion of the metal ion with an imino N-metal bond of 2 A 10,11,12 requires a 20~-30~ rotation of the bases which opens up the minor groove.
One hydrogen bond is retained in both base pairs so that rapid reformation of normal B-DNA without denaturation of the helix can occur on removal of the metal ion. The coordination geometry of the metal ion is distorted square planarwith the solvent providing the fourth ligand. The W-Vis spectrum of the Co2+
and Ni2+ isomers of M-DNA have peaks in the visible with ~ of 20 and 60 mol-1 cm-1 respectively; an observation which is consistent with this geometry 13 In the M-DNA duplex, the metal ion is buried within the helix and d-7~ bonding may occur with the aromatic bases above and below although we have no evidence for this.
The helix can be considered as a distorted member of the B-type farnily in agreement with the unremarkable CD spectrum 7. On average the metal-metal distance is 4 A
suggesting that electron transfer within the helix could occur efficiently.

This was investigated by preparing duplexes of 20 base pairs with fluorescein (the donor) and rhodamine (the acceptor) at opposite ends. Under conditions which favour B-DNA the fluorescence of the donor is quenched and the fluorescence of the acceptor is enhanced. This is an example of through space energy transfer (Forster resonance energy transfer or FRET) which has been well-documented in a number of different laboratories 14,15. The degree of quenching is due to dipole-dipole interactions and is distance dependent (1/r6); the value of 25% measured in our experiments is appropriate for this length of helix15. As shown in Figure 3a, the fluorescence intensity is relatively stable at pH 9 although at long times there is some loss due to photobleaching. On addition of Zn2+ the fluorescence is quenched up to 95% over a period of 1 hr, the rate of which mirrors the rate of formation of M-DNA under these conditions 7. As a control, the 20-mer duplex with only a fluorescein label shows a small decrease in intensity due to photobleaching as above (Figure 3a) . Similarly a mixture of two duplexes, one with fluorescein and one with rhodamine at one end, show minimal quenching either as B-DNA or M-DNA
(Table 1). Upon reformation of B-DNA by addition of an excess of EDTA after 4,000 sec, the quenching is rapidly reversed. These results are summarized in Table 1. The simplest explanation is that the excited electron on the fluorescein is rapidly transmitted down the M-DNA helix to the rhodamine; or, in other words, rapid andvery efficient electron transfer must be occurring. The Co2+ and Ni2+ isomers of M-DNA show quenching of the fluorescein by up to 95% even in the absence of the rhodamine acceptor (Table 1). One must conclude that the Co2+ and Ni2+
chromophores can themselves act as electron acceptors since the overlap with thefluorescein emmission spectrum is minimal.
The electron transfer in the Zn2+ isomer of M-DNA was investigated in a much longer helix of 54 base pairs with an estimated length of over 150A. The 54-mer also contained the recognition site for the D-site binding protein in the middle of the sequencel6. As shown in Figure 3b, there is no quenching due to FRET
because the fluorophores are now well separated. However, upon addition of Zn2+
to form M-DNA, the fluorescent intensity rapidly drops to 25% of the initial value.
In the presence of the D-site binding protein, the fluorescence intensity only drops slowly. However, as judged from the ethidium fluorescence assay 7, the majority of the DNA is still converted into M-DNA. Therefore, the DNA-binding protein is interrupting the flow of electrons by preventing interchelation of the metal ions to the central portion of the 54-mer. As a control, the D-site binding protein has no effect on the quenching of the 20-mer (Table 1). On addition of protease at 3000 sec, the protein is cleaved and the fluorescence intensity begins to drop, eventuallyreaching the minimum value of 25% as above. This experiment is a simple example of a bioreactive electronic switch.
M-DNA behaves as a molecular wire with a thickness of one atom. It is surrounded by a negatively-charged organic sheath which acts as an insulator andallows for its manipulation in electric and possibly magnetic fields. It is readily interconverted with B-DNA; therefore, the rules for cutting and splicing, and for the self-assembly of a variety of structures such as two and three-way junctions are well-documented 17,18. The binding of sequence-specific proteins can be manipulated to mimic electric switches and resistors. Even the manufacture of capacitors can beenvisaged by inserting a backbone-modified, uncharged stretch of M-DNA into a membrane. These are all ideal properties for the design of microelectronic circuits for the nanotechnology of the future.
Correspondence and requests for materials to J.S.L. at leejs~sask.usask.ca Acknowledgements. This work was funded by MRC by grants to J.S.L., L.J.T.D. and W.J.R., by HSURC by a post doctoral fellowship to P.A. and.by an MRC fellowship to L.T.

References 1. Dand~ker, P. J., Holn~in, R. E. & Barton, J. K. Science 275,1465-1468 (1997) .
2. Hall, D. B., Holn~in, R. E. & Barton, J. K. Nature 382, 731-735 (1996).
3. Arkin, M. R., Stemp, E. D. A., Holmlin, R. E., Barton, J. K., Hormann, A., Olson, E.
J. C. ~ Barbara, P. F. Science 273,475479 (1996).
4. Murphy, C. J., Arkin, M. R., Jenkins, Y., Ghatlia, N. D., Bossmann, S. H., Turro, N.
J. & Barton, J. K. Science 262,1025-1029 (1993).
5. Lewis, F. D., Wu, T., Zhang, Y., Letsinger, R. L., Greenfield, S. R., & Wasielewski, M. R.Science 277,673-676 (1997) .
6. Taubes, G. Science 275,1420-1421 (1997).
7. Lee, J. S., Latimer, L. J. P. & Reid, R. S. Bio~hem. Cell Biol. 71, 162-168 (1993).
8. Palecek, E. CRC Crit. Rev. Biochem. Mol. Biol. 26,151-226 (l99l).
9. Yagil, G. CRC Crit. Rev. Biochem. Mol. Biol. 26, 475-559 (1991).
10. Swaminathan, V. & Sundralingham, M. CRC Crit. Rev. Biochem. Mol.
Biol. 14, 245-336 (1979).
11. DeMeester, P., Goodgame, D. M. L., Skapski, A. C. & Warnke, Z. Biochem.
Biophys. Acta 324,301-303 (1973) 12. McGall, M. J. & Taylor, M. R.Biochem. Biophys. Acta 390,137-139 (1973) 13. Lever, A. B. P. "Inorganic Electronic Spectroscopy" (Elsevier, Amsterdam) (1988).
14. Cheung, H. C. in "Topics in Fluorescence Spectroscopy" pp 128-171, ed. Lakowicz, J. R. (Plenum, New York) (1991).
15. Clegg, R. M. Methods in Enzymology 211,353-371 (1992).
16. Roesler, W. J., McFie, P. J. & Dauvin, C. J. Biol. Chem. 267, 21235-21243 (1992).
17. Lilley, D. M. J. & Clegg, R. M. Ann. Rev. Biophys. Biomol. Str. 22, 299-328 (1993).
18. Seeman, N. C. & Kallenbach, N. R.Ann. Re~. Biophys. Biomol. Str. 23, 53-86 (1994).
19. Brunger, A. T. X-PLOR Manual, Version 3.1 (Yale University Press, New Haven USA (1993).

Figure Legends Figure 1. Release of protons on formation of M-DNA. Upon addition of NiCl2 protons are released and KOH was added (left axis) to maintain the pH at 8.5. After each addition 10 Ill was removed to assess the formation of M-DNA by the ethidium fluorescence assay7 (right axis). The experiment was performed in a 10 mL volume, with 1.1 rnM in base pairs of calf thymus DNA. The DNA was dialyzed against water and sheared by passing through a 30 gauge needle five times. Arrow (a) indicates the point at which M-DNA formation began. This lag phase is proportional to the DNA concentration (data not shown) and is presumably due to the initial binding of the metal ion to the outside of the helix. Arrow (b) indicates the point at which 1.1 mM of H+ had been released, beyond which precipitation was observed.

Figure 2. Modelled structure of M-DNA. (a) Stereo diagram of a mixed-sequence 12base pair helix. Zinc ions are shown as green spheres and are buried in the centre of the helix. Overall, the structure is similar to that of B-DNA with about 11 basepairs/turn. The model was generated from a canonical ~DNA helix by replacing theimino protons of guanine and thymine with zinc ions and then empirical energy minimization was performed with X-PLORl9. Parameters describing the coordination of Zn2+ were included in the minimization together with a water as the fourth ligand. (b) G~ and A-T base pairs from the modelled structure. Hydrogen bonds and interactions between Zn2+ and its coordinating groups are shown as dotted lines. To accommodate the metal ion the base pairs open towards the minorgroove with an additional shearing of the purine towards the same groove. In the G~ base pair, the coordinated water molecule mediates the interaction and helps to neutralize the electrostatic repulsion between the metal ion and the 2-amino group of guanine. In the isomorphous A-T base pair the water molecule occupies a sirnilar position.
Figure 3. The fluorescence of fluorescein-labelled oligonucleotides during the formation of M-DNA (see Table 1 for the sequences of the 20-mer and 54-mer) (a) Effect of Zn2+ on the 20-mer duplex. (i) Fl-20-mer duplex without Zn2+; (ii) Fl-20-mer duplex with Zn2+; (iii) Fl-20-mer-Rh duplex in the absence of Zn2+; (iv) F1-20-mer-Rh duplex in the presence of Zn2+; (v) addition of EDTA after the formation of M-DNA. (b) Effect of Zn2+ on the 54-mer duplex. (i) Fl-54-mer-Rh with D-site binding protein (1~g/ml) (the site is located at the centre of the 54-mer duplex) in the presence of Zn2+; (ii) addition of proteinase K (50~Lg/ml) after 3,000 sec; (iii) F1-54-mer-Rh duplex with Zn2+. The experiments were performed in 20mM NaBO3 buffer, pH 9.0 at 20 ~C with 10mM NaCI and 1 mM Zn2+ as appropriate.
Fluorescence intensities are normalized with respect to the fluorescence intensity of the Fl-20-mer-duplex either in the absence or presence of Zn2+.

Table 1. Normalized Fluorescence of the Fluorescein-labelled oligonucleotides Oligonucleotide Treatment Fluorescence Fl-20-mer duplex ~--.. +zn2+ 0.98 +Zn2+ at pH 8.0 0.92 Fl-20-mer single strand ---- 0.87 Fl-20-mer duplex + ~-- 0.97 Rh-20-mer duplex Fl-20-mer-Rh duplex ---- 0.73 .. +zn2+ 0.05 +Zn2+ + EDTA 0.87 +Zn2+ at pH 8.0 0.92 +Co2+ 0.05 +Co2+ +EDTA 0.7 .. +Ni2+ 0.06 " +Ni2+ +EDTA 0-7 .. +Mg2+ 0.83 " +D-site binding protein 0.06 +zn2+
Fl-54-mer-Rh "
.~ +zn2+ 0.21 All experiments were performed in 20mM NaBO3 buffer, pH 9.0 (or 20 mM
Tris pH 8.0) with 10mM NaCl at 20 ~C and 1 mM Zn2+ or 0.2 mM Co2+ or 0.2 mM Ni2+ or 2 mM EDTA as appropriate. Excitation was at 490 nrn with emmision at 520 nm. Fluorescence intensities are normalized with respect to the fluorescence intensity of the Fl-20-mer-duplex either in the absence or presence of Zn2+ and were measured after 3,000 sec. Sequences and nomenclature:
The oligonucleotides were labelled 5' with Fluorescein (Fl~ or Rhodamine (Rh) and were obtained from the Calgary regional DNA synthesis laboratory.
Fl-20-mer:-F1-5'-d(GTC ACG ATG GCC CAG TAG TT) Rh-20-mer:-Rh-5'-d(AAC TAC TGG GCC ATC GTG AC) and the same unlabelled sequence was used to produce the Fl-20-mer-duplex.
Fl-54-mer:-Fl-5'-d(GCT ATG ATC CAA AGG CCG GCC CCT TAC GTC AGA
GGC GAG CCT CCA GGT CCA GCT) (The ~site is underlined) Rh-54m:-Rh-5'-d(AGC TGG ACC TGG AGG CTC GCC TCT GAC GTA AGG
GGC CGG CCT TTG GAT CAT AGC) and the same unlabelled sequence was used to produce the Fl-54-mer duplex.

2.0 .o~4 ~ ~ 100 1.5 -- ~ O
~-1.0- ~_ 50 a ~ 25 ~
v~a 0.000~ ' ' O
0.0 0.5 1.0 1.5 2.0 [NiCI 2]~ (mM) H\ I =\
/N H-- - -O ~N_ R

R O

~ 2~, H ~1~
~_</~ --H N~N~ R

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H

~ ,.

~ ~b =~c (a) . ~ (i) ~ ~ ~ (V) 0.6 - ~ i) _ 0.4 - \
C (iv) \
0.2 - \

~. I ~ I

C ~ ~_ (i) (b) - 0.8 - I ~
' I ~ (ii) Z 0.6 - ~ \
I (iii) \
0.4 - ~ \

-O

Time (sec~nd~) A cooper~tive conformational change in duplex DNA induced by Zn2+ and other divalent metal ions IEREMY S. LEE ~ND LAUR~ J. P. LATIMER
..~ Or Bloc' r J~, University of Soskat~hewan, Sn~ a,., Sask., C~nada S7N OW0 AND
R. STEPHE~ REID
Dt, , Jl of Chemisrry, Universi~y of S ' ~ ~Ic~.. ., Sasko~oon, Sosk., Conado S7N OW0 Received November 13, 1992 LEE, J. S., LATlMER, L. J. P., and RElD, R. S. 1993. A COO~...I;.~COnrOrmatjOnal change in duplex DNA induced by Zn2~ and other divalent melal ions. ~iochem. Cell Biol. 71: 162-168.
Zn2~ and some othcr divalcnt metal ions bind to duplex DNA at pHs above 8 and cause ~ confG- -' change.
This new structure does not bind ethidium, allowing the dc~.'.,, - of a rapid r ~ - assay. All duplex DNAs, regardless of sequence or G-C content, can form this structure. The rate of formation shows a strong d~, ~
on ~ , pH, and Zn2~ r .,Lon, at 20~C, I mM Zn2~, and pH 8.6 the ~' is half complete in 30 min. Addition of EDTA causes rapid reversion to ~B~ DNA, showing that the ncw conformation retains two strands that are ~ " ' Unlike the ultraviolet or circular dichroism speara, the nucl~ar magnetic resonance spectrum was informative since thc imino protons of both A ~ T and G C base pairs are lost upon addition of a 5~ ;C amount of Zn2~ . The pitch of the helix was estimated from gel clc.l.oph ~;a of circular DNAs in the presence of Zn2~ and it contains at least 5q. fewer base pairs per turn than ~B' DNA. The transformation is coo~.at;~e and shows hysteresis, suggesting that this is a distinct structure and not simply a minor vuiant of ~B~ DNA. It is proposed to call this ncw struaure 'M' DNA because of the intimate i...ol.. of metal ions.
Ke,v words: DNA . ~o., COv~ dti.~ trulsition, ahidium binding, divalent maal ions, proton nuclear magneaic LEE, J. S., L~TIMER, L. 1. P., et RElD, R. S. 1993. A coOp~ confor ?I ' change in duplex DNA induced by ZD2~ and other divalent metal ions. Biochem. Cell B;Ol.71:162-168.
Lc zinc (Zn2 ~ ) et d'autres ions l ~ " ,_ divalents se lient à l'ADN b;c~~' - e lorsque le pH est supérieur à 8 et ils p.- ' un ' _ de conformation. Cette nouvelle struaurc ne lie pas l'ethidium, ce qui a permis la mise au point d'une méthode de dosage rapide par lluv-~ ~ Tous Ies ADN bir~;- ., peuvent forma cette struc-ture peu impone lcur sequence ou leur contenu en G ~ C. La vitesse de ce c'~ _ de conformation est fonement ~;F ' edelai .' _ ~,dupHetdela,;~n~ - a dcZn2~;1a ' - - n. fft àmoitiécomplétéeen30min a 20~C, Zn2~ I mM et pH 8,6. L~addition d'EDTA entral'ne un retour rapide à la conformation '8' de I'ADN, ce gui démontre que la nouvelle conformation est formée des dew~ chaines a~ni~Ja~ "'' CO..nail. aux speares ultraviolct ou de ~I;.h.,: - circulaire, le spectre de résonance ~ L. nucléaire est informatif puisque les protons des e~ ~ _, imino des deux paires de bases A T a G C sont perdus lors de I'addition d'une quantité s~ e d~ Zn2 ~ . L'~ - du pas de I'hélice par cl.~llvt~hv.~se en gel d'ADN circulaires en presence de Zn2~ indique que Ie nombre de paires de bases par tour qu'elle contient est au moins 5q inferieur à celui de l'ADN de conformation 'B'. Cette transformation est ~OVP~aI;~eeteIIe montre une hystéresis, ce qui sug8ère que cette conformation est une structure distincte et non seulement une variante mineure de la conformation ~B' de I'ADN. Nous proposons de nom-mcr cette nouvelle structure, I'ADN de conformation ~M', à cause du rôle esscntiel d'ions, ~
Mors clés: conformation de I~ADN, transition .oope. ~ , liaison d'éthidium, ions ': "; divaknts, résonance ~, -, nucléaire.
Traduit par la rédactionl ~ I ~d ~I ~r Divalent cations are perhaps more interesting because a The binding of metal ions to DNA has been studied exten- series can be written in decreasing order of DNA stabiliza-sivdy for nearly 40 years. In genaal, cations that bind tion i.e., {Mg, Co, Ni, Mn, Zn, Cd, Cu~ (Eichorn 1962;
primarily to the phosph~e backbone will stabilize the duplex Eichorn and Shin 1968). Thus Mg2+ increases the Tm at all conformation, whereas those that bind to the bases will tend ~o~ .àlions, wher~as sufficientlY high col-ntl alions of to denaturc the duplex. Thcse effects are readily demon- Cu2+ will lead to ,i al~,lation of the duplex at room tem-strated with thermal denaturation profiles ( Tm measure- perature (Eichorn and Shin 1968). This series also correlates ments). Thus most l-.ono-a' cations such as Na + with thc ability of the ions to bind to the bascs (Hodgson stabilizc thc duplcx and there is about a 12~C increase in 1977; Sw~nin~h~n and Sullda.àli~.,,l.all. 1979).
Tm for cach l~fold increase in conc~ lion (Marrnur ant As well as influencing thc hclix to coil l-an~ilion, cations Doty 1962). An important cxception is Ag +, which binds are also involved in pl- e several other structural tran-tightly to thc bases and therefore decreases the Tm (Guay sitions and ~ nc First, Mg2+ and pol~ ~ both and B~ 1979)- Similarly multivalent ions, particu- favour the ~ ion of pyr ~ pur DNAs to triplexes (Htun larly thc pol~ , arc very cffectivc duplex stabilizers. and Dahlberg 1988; Maher e~ al. 1990; Haner and Dervan A8BRE~IIATIONS: pyr, ~" " ~ pur, purinc, NMR, nucl~ar 1990;Hampeletal. 1991).Thiseffectcanbequitedramatic, ma~a;ic r~ . MES, ~ ' - .-' '' acid- CHES since the presence of spermine can obviate thc rC~ui~ nl Ie ~ . '', -acid;CD,circulardichroisrn;DSS, for low pH in the formation of pyr-pur-pyr triplexes.
3~(n '~' 'yl)-l-p.o~~ 'fonic acid. Second, L~lca.ing the ionic strcngth pa~ la~ly with c~ ~ Imvrm~ c~

CA 02218443 l99X-10-26 LEr ET ~L 163 multivalent cations c.. ~l.. agcs thc formation of Icft-handed 80 'Z' DNA from dl,propriatc alternating pyr-pur s~q. n~
(Rich e~ ol. 1984; Palecck 1991). Third, a r~ ca~d DNA ~ A
structur~ which occurs in (C-A),~ tracts and rcquircs sper- ~ ~Q
mine for stability has bcen dcscribcd recently (Timsit et ol. I ~ pH=6 5 1991). It seems that all thrce of thcse structurcs can occur 70 - ¦ ~ ~
undcr p~ aiO~ cC~ nc and thcy may have ,~ x . ~r,t ~ ~
bi -'~, ' roles (Rich el al. 1984; Lee et at. 1987). Mctal ions ' ~ \
are also found in some DNA-binding agents such as cis- ~ pH=9 plalinum t~ ~c;-- and Fe(lI)-EDTA (Rosenbcrg et al. 'o1969; D'Andrea and Haseltine 1978; Latham and Cech 60 1989), and many DNA-binding proteins rcquire specific 0 100 200300 400 500 600 cations for activity (Mildvan and Loeb 1979). For examplc, Zinc concenl,dllon (IlM) the 3 ',5 ' -cxonuclcase of DNA polymerasc I from FIG I EffectofZnl~onthcmeltingi a~ (Tm)ofcalf F~ . 7 CO/; has two metal ions in thc actlvc slt~ (B~sc ~hymus DNA at pH 6.S (_) or 9 (~).
and Steitz 199t).
In this paper we describe the discovery and p. Cli~ ar~ Once formcd. thc ncw conformation is stable in this buffer, but characterization of a novcl DNA conformaIIon In which th~ Zn2~ c~r - ,~ion is too low to allow the ' - on of 'B' divalent metal ions play a very Intlmate rolc- Thls lln~ of DNA Addition of 2 rnM EDTA to thc ethidium-zinc buffer research arose quite fortuitously. Originally, Tm mcasure- rapidiy chclat~s th~ Zn2~ and conv~rts it to the cquivalcnt of ~he ments wcre being performed to investigate thc cffects of standard ~thidium nUO- . ~e assay containing 0.1 mM EDTA, Zn2+ and Mg2+ on the stability of pur-pur-pyr triplcxes but no Zn2~, as described earlier ~Morgan er ol. 1979). With (Kohwi and Kohwi-Shieem~)su 1988; Lyamichev et al. excitation at 525 nm and cmission at 600 nm, 0.5 1lg of duplcx calf 1991). It was dccided to use a pH of 8 ot greater to prevent thymus DNA gives 70 fl n.. - units and thc scalc is linear in intcrferencc from triplexes of th~ pyr-pur-pyr type (Lce thc range of 0-200 ~ ~;cs units.
et ol. 1984). It soon became apparent that a different struc- Kinel~cs ture which was dcpPn~l~r~ on Zn2~, but not Mg2+, was Standardrcaaionswercpcrformedat20~Cin300~Lofasolu-being formed. This new struaurc was availablc to all DNA tion containing 75 ~-M DNA, 15 mM NaCI, 10 mM Tris-HCI
duplexcs rc~Sa. .~ of sequcnce. Furthermore, it did not (pH 8) (from the DNA solution), and 20 mM CHES (pH 9). The bind ahidium, so that a rapid fluorescence assay WâS quickly final pH was found to bc 8.5. Other buffers uscd were 20 mM
developed (Morgan e~ al. 1979). In this wây, most of the CHES (pH 9.5) (final pH 9.0), 20 mM Tris-HCI (pH 8.0) (final pleL,~ a.~ charaacrization of this structure could be pH 8.0), and 20 mM MES (pH 6.5) (final pH ~.0). Thesc solutions pcrformed . Later, NMR studies confirmcd that a Zn 2 ~ w~r~ ~ with ZnC12, which was always added last. At atom was replacing the imino proton common to both G ~ C vari~Us timcs 3~.L al~qUbcutfSfcr and tPhc rcading was takcn within and A-T base pa~rs. 5 min.
Malerials snd me~hods Circular Dichroism ~ucleic ocids CD spcctra werc kindly performed by A.R. Morgan ~F.' -Synthctic duplcx DNA was preparcd by ~~ of an appro- in a buffer of 20 mM sodium borate (pH 9.0) with or without priate templatc with E. coli or ~f;.,~cocc~.s lu~eus DNA pOIy. I mM ZnC12. Thc calf th~nus DNA ~ ~ r 1. was 150 ~.M.
mert, sc I as described previously (Evans et ol. 1982). Open circular Nuclear Magnetic Resonance pUCI9 was prepared by r-irradiation with a dose of 8 x 10 rad Spcara werc measured on a Bruker Am 300 , . .d~
(I rad = 10 mGy). Relaxed pUCI9 was prepared by inr bmi-. magnet s~ a at a frequency of 300.13 MHz. Chemical with 2 units/~g of calf thymus t, p 2 ~ ~ ~ I (BRL) for I h, shifts ar~ quoted in ppm relative to DSS as an internal standard followcd by deprot~ with phenol. Calf thymus DNA and with a probe ~- , c of 2S~C. Samplc volume was 500 I-L con-bacterial DNAs were purchased from Sigma. All DNAs were taining 5 mM DNA (F~s ' ~) in 90qo H2O- lOqo D20. The dialysed into a buffa of 10 mM Tris-HCI (pH 8) and stored frozcn Hore binomial pulse scquence ' 1331 ' was employed for water sup-at -20Cc. pression(Horel983).Aninterpulsedelayofl85~s~o-,~r.,d g Thermal ~1 ~ _ io prof les to an excitation maximum at 13.5 ppm was used. Repetition times ~ - - were made on a Gilford 600 sp~ctro- was 5.2 s. Between 1000 and 50Q0 transients were a - ' ~ for phntc r equipped with a thermo~"o~l_ in either each CO~l~Im~liOfl of ZnC12 depending on the magnitude of the (a) 20 mM sodium boraIe (pH 9.0) or (b) ~0 mM MES (pH 6.5) peaks in the aromatic reg~on.
,, ~.1 with ZnC12. At pH 9 the solubility limit of ZnC12 Agarose gel ~ rOp,~
is about 500 ~-M increasing to about 5 mM at pH 8.S. Addition Gels (I qo agarose) were run at 60 v for 5 h in a buffer of 20 mM
of 15 mM NaCI to some buffers also incr~as~s the solublllty of sodium borat~ (pH 9) ~u~F'~ '~ with up to 0-5 mM ZnC17 ZnC12. Thus, in general, great care had to be t?ken in making up The plasmid pUCI9 (0.5 ~.g) was incubated for 30 min with the buffers a~ - 2 Zn-~ to prevent ~)r~ a. It should also ~, ., atiol~ of ZnC12 before addition to the gel.
be noted that DNA itself acts as a chelating agent and therefore can increase the apparent solubility of ZnC12 quite conside.ibl~. Resulls Figure I shows the effect of i.~ ashlg Zn2+ c~,..c~ ,a All fluorescence - I - were performed on a Turner modd 430 ~c.t.~,f' - ~ :u in a buffer con~ 5 mM Tris- tions on the Tm of calf thymus DNA at pH 6.5 and 9. At HCI (pH 8.0), 200~M ZnC12 and 0.S ~-i/mL of ethidium pH 6.5, the mqYjmllm inctease in Tm was only 3~C. This (ethidium-zinc buffa). This buf;er was chosen to take advantage agrees with earlier work by Eichorn's group performed at of the hysteresis in the d - - n.~ (see below). Iow pH, which had sugg~s~ed that Zn2~ was mostly 164 BIOCHEM. CELL BIOL, VOL. 71, 1993 120 ~ ~ 120 ~ ~ ~ ' ~ ' (A) Zlnc cDl,esl at'~n ~ ~ (~) ph '~ 1 00C~ - l oo~
80 ~ ~ ~02~M' 80 ~--~7 . 60'~ u\ ~ 60 'o aos,ru' 40 ~ ~ ~
20 ~ A 20 ~ ~5 ~G 05~ o '~? ~ . Q 9 .

. , 120 ~ ~ ~ ' ~ ~ 120 ~ ~
(C) Te.. , Iralul~ L (D) Metal lons 100D 100~ , o u~u~
80 ~ ' 80 ~o~, ' 60 ~e 20~ It U207~ 40 ~
0 ' ' ' ' ' ' ' ' ' ' ' ' " 0~--~
~ 50 1 00 1 50 0 20 40 60 80 Time (min) Time (min) FIG.2. (A) Thc d ~, at different Zn2+ con.~.. t,~ - and pH 8.5 as mcasurcd by loss of ethidium fl e : ~, 200 IIM;
', 500 IIM; ~, I mM; _, 5 mM. (B) Th~ at differcnt pHs and I mM Zn2~ as m~asurcd by loss of ethidium lluor~ -~
-, pH 7; ~, pH 8; ~, pH 8.5; ., pH 9. (C) Th~ ~' - ). at differ~nt ~ ~ albl.~ and I mM Zn2~ and pH 8.5 as measured by hss of ethidium fluorc~,c.. ~e. o, 0~C; o, 20~C; 3, 37~C. (D) Th~ at pH 8 5 with I mM of various divalent metal ions.
~, Mg2+; ~ Mn2+; ~, Cu2+; ~ Zn2+; ~ Ni2~; ~ Co2+

destabilizing (Eichorn and Shin 1968). On the other hand On the other hand, if poly[d(G-A)] was added to a solution at high pH, Zn2+ stabilized the DNA duplex by as much of polyld(T-C)] under these con~litions (i.e., with EDTA), as 12~C. The effect of pH was most unP~cte~l but sug- no [luo-~cace could be detected (data not shown). Previous gested that some form of specific metal ion complex or workhasd~..lor~lraledthatthisabsenceofr~ndlu-dtionwas structural rearrangPmPnt was occurring. In either event it due to the very dilute DNA :r ations and the low ionic seemed rP~con~k to propose that ethidium might not bind strength of the nuolcs;..lcc assay buffers (Morgan et al.
to the new structure. This led to the devcloF of a rapid 1979). Overall, this is an ~ cl~ revealing result because n ~ ~ence assay, since indeed the new conformation does it shows that this ~ n;on does not involve separation not enhance the lluol.sc ~: of ethidium of the strands as occurs in dc.,~lu.alion or in triplex for-lt was found by trial and error that pH of 8 and 200 ~lM mation (I.ee et ol. 1984).
Zn2~ were convenient for tluo,~scen.e ~..ea~u-~.n...ts, since The effect of pH is investigated in Fig. 2B. Above pH 8.5 the new structure would not form under these conditions; the riismlJ~ation occurred rapidly, whereas below pH 8 very but once formed, it remained stable for at least 30 min. Iittle transformation was detectable. Again addition of Thus, the ~-I.asu~.d fluol~sccll.c could be used to estimate EDTA increased the fluorescence back to 100qo. Therefore, accurately the amount of unmodified DNA in a sample even at pH 9 the observed fluorescence loss was not due to undergoing the transformation. denaturation of the DNA. The rate of formation was Tbe kinetics of formation as a function of the Zn2+ con- increased with increasing t~l~.pe.alulc (Fig. 2C). However, ccntration at pH 8.5 is shown in Fig. 2A. At co---e.-tl.-tions even at 0~C the conversion would go to co "1:~ on above I mM Zn2+, the conversion was very rapid, while eventuaUy.
below SOO ~M formation of the new structure did not go Several other kinetic CA~.illllts were performed to to co F ' M 3 n even after prolonged incubation. This narrow elucidate the new structure. The ~licmnr~ n of calf thymus conc~ la~ion range is suggestive of a coo~lalive transition. DNA was measured in the presence of hl;ltd~;ug concen-Again this is typical of other conformational changes such trations of ethidium. At I mM ethidium the conversion was as 'Z' DNA (Rich et al. 1984). Another attractive feature c~ Iy inhibited (data not shown). Therefore, the lack of this fluorescence assay is that e.~.h..-.lls can be per- of ethidiumfluorclc~..cewasmostlikelYduetoweakbind-formed directly in the ethidium buffer solution. For exam- ing co...ydrcd with 'B' DNA. Thc ~licn~ ion of poly-plc,additionofanexcessofEDTArestoredthefluo.eî.~.l.e ld(T~)]-polyld(G-A)l was measured in the presence of immr~li?trly (within I or 2 s) to lOOqo of the value that a~ ion~l polyld(T~)] and polyld(G-A)I (data not shown).
would be expected for the buffer in the absence of Zn2+. Neither had a significant cffect on the rate of formation LEE ET ~L. 165 again, s~gR-sting a two-s~randed structure. A-iso consistent 120 - ' ' ' ' ' with this result was the lack of a significant concentration 100O (A) ~aeterlal DNA~
effect. For example, lowering the DNA conce.ll-dtion by 10-fold did not change the rate of transformation, ~3 ' 2 80 an intra-, rather than an inter-mol~ular .c~. ~. ~--e~ ~ The effect of base composition and sequence on the rate was 60 investigated (Fig. 3). As shown in Fig. 3A bacterial DNAs ranging from 72~70 G + C (M. Iuteus), through 50qo G + C
(E. coli), to 32qo G + C (Clostridium perfringens), all 20 ' J- ~ed atequivalent rates. Itwastherefore l-nPYpected , ~ , ~
to find that synthetic DNAs showed large differences ~0 20 40 60 80 (Fig. 3B). Under these conditions poly[d(A-T)] was l, whereas poly(dA) - poly(dT) readjly dicmu~20 and polyId(G-C)l was intermediate in rate; poly[d~T-G)]~ ) Synth-tic DNAr polyld(C-A)I appeared to be panicularly willing to ~ 100~, ~licm~ , 'Finally, synth~tic DNAs con~ining modified <~ 80 G Cl bases are shown in Fig. 3C. Unlike polyld(A-T)I, both polyld(A-U)I and polyld(z7A-T)I formed the new structure ~ 60 ~--slowly. Other DNAs containing 1, m6A, and z'A all o ~ ~,~
~u~ed rapidly. These last two modifications were ~ 40 imponant because they eliminated the possibility that ~L 20 ~ L
Hoogsteen pairing was involved in the structure.
Other divalent metal ions were also evaluated (Fig. 2D). ~ ' ' ~ ' Ni2+ and Co2+ behaved likeZn2' but Mg2+, Mn2+, and ~ 20 40 60 80 Cu2~ wcre ineffcctive. Thus there appeared to be no cor-relation between the ability to bind to the bases and the 120 ~
ability to form this new structure. Ag+ was also tested, 100~ (C) Modined DNAs since it is known to form stable co 1" ~ with the bases (Guay and E~eauch~F 1979). It was found that under the 80 con~ ionc of Fig. 2D, addition of I mM Ag~ immer~ y r~
led to a 100qo loss of fluorescence. However, addition of 60 ~r~
EDTA caused no increase in fluorescence, demonstrating that the DNA had been denatured. This served as an excel-lent control to contrast with the effect of Zn2+. 20 .
Spectroscopic characterization of this structure was ' ~
invfftigated The UV absorption spectrum showed little ~o 20 40 60 80 change. Compared with ordinary 'B' DNA, there was a Time (min) small h~luc,..~ (about 10%) at 260 nrn, but in general this was insufficient to allow easy detection or analysis (data FIG.3. E~fect of DNA sequence on thc rate of the ~" ' not shown). The CD spectrum was compared to that of 'B' (ANA - ~ ! """"5 'lu~ s DNA ~Bj~Sy'nthaic DNAs- O poly-DNA ffig. 4). Sul~ inglj, for a structure that did not bind [d(A~ , polyld(G C)l; ~, poly[d(T-T C)l-poly[d(G-A-A)I; c, ethidium, the spectrum showed only small chang~s- In poly[d(T C-C)I poly[d(G-G-A)l; ~. poly(dA) poly(dT); ~. Poly-general the depression in intensity of both the positive and [d(T-G)I poly[d(C-A)l. (C) Modified DNAs: ~, poly[d(A-U)I; ~, negative bands was similar to that of DNA in the presence poly[d(z7A-T)I; ~, poly[d(m6A-T)I; o, poly[d(T-C)I-poly-of high concc.-lraIions of cesium (Zimmer and Luck 1974; [d(G-m6A)I; ~, poly[d(T C)l-poly[d(l-A)l; ~, poly[d(T4)1-poly-Chan et a/. 1979). [d(G-z~A)I.
The NMR spectra of d(T-G)~5-d(C-A)~5 in the absence and presence of Zn2' is shown in Fig. 5. The imino pro- to an imino proton which is adjacent to a metal ion.
tons of both the A-T and G-C base pairs at 13.4 and If thenewstructurecontainsaZn2+ atomforeverybase 12.3 ppm, respectively (Sklenar and Feigon 1990), appeared pair, the winding of the heli~ might be expected to be rather abscnt in the spectrum of the new structure (Fig. 5C). Large different. Thus the final series of e,~. ile.lIs were designed changes were not observed in the rest of the spectrum. At to investigate the topal~ PI properties of circular DNAs intermediate Zn2+ COIlCe"~rdlions (Fig. 5B) the peaks at in the presence of Zn2+. Supercoiled, relaxed, and nicked high field were d.";e~cd and a small peak appeared at plasmid DNA was electrophorcseJ on Iqo agarose gels at 12.9 ppm. ' pH 9 wjth in.l~ ' G con.~ lions of Zn2+ (Fig. 7). Elec-Titration of the imino protons is shown in Fig. 6. The I-opho..;.is under these cor-1:lionc led to more diffuse bands A-T imino proton was lost before the G-C proton which may with some smearing . - - cd with clc.IIophorcsis at pH 8.
explain some of the sequence effects described above. Com- The control with no znf+ showed that relaxed and nicked pl~te loss of the signal occurred at about 2.4 mM Zn2~ DNAs (both l-.ono...c. and dimer plasmids) had thc same which is equivalent to one metal ion per base pair. This is mobility, and some t"F ~ , wcre also di~c~ in the co~ en~ with every imino proton being replaced by Zn2+ relaxed sample. At 0.5 mM Zn2+, on the other hand, the or a rapid exchange process. The proton at 12.9 ppm that relaxed and ,~ DNAs had the same .,.ot "- and appeared at intermediate Zn2+ conc~.lIl.-tions may be due no tOpO;s~,..lc" could be seen in the relaxed DNA. As well, 166 BIOCHEM. CELL CIOL. VOL. 71, 1993 ~0 220 2~0 260 ~B0 3 WAVELENGTH (nm) FIG. 4. Circular dichroism Or calf thymus DNA at pH 9.0 in thc abscnce ( ~ - ) or presence of l mM Zn lD~

0.8-0~ - o OL - _ I

o ' ~ ~ 0.2- ~ -'. t. h ~ W, ~1 ~ 8 0.0 ' ~ ' ' (A) ~) (C) 0 0 0.5 1.0 1.5 2.0 2.5 Zinc Concentr~tion tn~M) .0 12.0 13.0 12.0 13.012.0 FIG. 6. ~J~ integrals v~sus ZD2 , . for th~
ppm imino protons of 5 mM d(T-G)l,-d(C-A)". ~, 12.4 ppm; o, FIG. 5. Irnino p~oton NMR of d(T-G),5-d(C-A),5 at pH 9.0 13.4 ppm; ~, 12.9 ppm. This l_st signal is only observed at inter-with (A) 0, (8) 1.2, and (C) 2.4 mM Zn2~. mediate Zn2~ ~c ~i -the presence of Zn2+ tended to decrcase the sharpness of Discussion the bands. The sudden change in mobilities at 0.4 mM again It is clear that the new structure does not represent a minor demonstrated that the transition was coop~.a~Bc. Here the structural variation along the continuum of 'B' DNA struc-mobility of the relaxed sample, particularly the relaxed tures. First, it does not bind ethidium well, as this drug dimer, was interrnediate between the nicked and supercoiled inhibits the dismutation. 'A' and 'B' DNA, as well as RNA
DNAs. Thus, the dismutation induced negative ~u~.coils and even some triplexes, bind ethidium. Second, it demon-and the helix contains fewer base pairs per turn than 'B' strates hysteresis since it remains stable under ~on~ onc in DNA. Attempts to measure the overwinding accurately by which it will not form ffig. 2). In other words, the structure two~; n~ n~ onq~ cl~I~opho~csis (Haniford and Pulleyblank formed under a certain set of con~itionC depends on the 1983; Yagil l991) were u -ncc~rul, because the presence route taken to achieve those cond;t;ons. This manif~station of Zn2 always inl-odu.ed a level of smearing which of hysteresis is often seen in other ~lluaulal transitions of obscured the t . pois ~. However, from Fig. 7 it can be DNA (Rich et ol. 1984; Lee er al. 1984; Hampel et a/. 1991).
cstimated that the new structure contains at least 5~o fewer Third, the transition is coopc.ali~c as judged from the base pairs per turn than 'B' DNA. It is also apparent from effects of iuer ~ e Zn2~ conce.-l-aIions in the kinaic Fig. 7 that there is an increased mobility and that the separa- t,~.h-IS (Fig. 2) and in the agarose gel cI~rophOlctic tion of nicked and supercoiled species is reduced. Both of studies ffig. 7). None of these p-o~. Iic~ would be expected these faas would be q-~icipat~d if the new helix is shorter of a minor variant of duplex DNA, but are more typical and more compact than in 'B' DNA (Mickel et al. 1977). of the behaviour of a distinct structure such as 'Z' DNA.

LEE ET AL. 167 OmM0.2 mM 0.3mM 0.4 mM Q5 mM
~ 1 2 3 ~ U 1 2 3 ~ ~ 1 2 a ~ ~ 1 2 3 ~ ~ 1 2 3 .t FIG 7. Agarosc gel ek.ll~ph~,.c~ at pH 9.0 with 0, 0.2, 0.3, 0.4, and 0.5 mM Zn2+. M, ~ molecular weight markers; lane I, ccc plasmid; lanc 2, topn'S_ aae-relal~ed plasmid; lane 3, oc plasmid. In lanes I and 3, at 0 and 0.5 mM Zn2+, 'M' and 'D' show the position of the plasmid monomer and dimer, lea~~
It is proposed to call this new structure 'M' DNA because than the unmodified polymers; the relevant pKas are U
of the intimate h~vol~e.. l.. ll of metal ions. (9.3) and 1(8.8). The metal ions which favour 'M' DNA;
Many potential structures which might be assigned to 'M' that is Zn2+, Ni2+, and Co'+ all have ionic radii of about DNA can be eliminated on the basis of a few key observa- 0.70 A or less, whereas Mn2+ and Cu2+ which are innef-tions. First, rapid restoration of normal duplex DNA upon fective have radii of 0.80 and 0.92 A (I A = o.l nm), addition of EDTA to the ethidium fluorescence buffer lea~ l),(CottonandWilkinsonl966).Theoneexception cannot be eYp!qin~d by denaturation, a triplex structure, or is Mg2+ with an ionic radius of 0.65 A, but Mg2+ does one Co~ ;..g parallel strands. Second, since 'M' DNA can not form stable complexes with nitrogen bases. Ag + was be formed by all DNAs regardless of sequence, then A must of interest because it is known to form stable ~opl.,~ with still pair with T and G with C. Third, Hoogsteen or other the bases in which the imino protons are lost (Guay and types of hydrogen bonding can be ~limin~ted because m6A Be~ homr 1979). However, it will not form 'M' DNA, but and z7A do not inhibit the tlicmut~ n. Therefore, these instead denatures the DNA. Its ionic radius is l.l3 A, which results imply that some features of the usual Watson-Crick may be too large to be accommo~ ed within the helix. Spe-base pairs are retained in the structure of 'M' DNA. Fourth, cific cation effects have also been noted in the formation the NMR clearly shows that the signal for the imino protons of tetraplex structures where again a metal ion is bound has been elimin~ed and that there is one Zn2+ for each tightly to the bases (Lee 1990; Zahler et al. 1991).
base pair. This can be interpreted either as a replacement Binding of the Zn + undoubtedly leads to distortion of of the imino protons by Zn2+ or the formation of a com- the helix. For example, imino-Zn2+-imino bonds are plex which allows for their rapid exchange with solvent. usually about 4 A compared with 3 A for the Watson-Since Zn2+ usually forms tetrahedal complexes, several Crick base pair (sw~min~th~n and Sundralingham 1979).
functional groups on the bases or the sugar-phosphate The presence of the metal ion would also reduce the total backbone are probably involved. charge on the helix. This, in turn, would tend to compact Whatever the structure, the tight association of Zn2+ the heliY~ and decrease the number of base pairs per turn as with every base pair explains many of îhe properties of ~M' was observed in Fig. 7. Further details of the structure of DNA. For example, ethidium does not intercalate because ~M' DNA will have to await X-ray crystallographic or two-of charge repulsion; triplexes containing C-G-C+ base dimensional NMR studies.
triads will not accommodate ethidium for the same reason Zn2+ is one of the few metal ions which can coordinate (Morgan et al. 1979; Lee et al. 1984; Scaria and Shafer 1991). well to both oxygen and nitrogen. Therefore, together with Addition of EDTA rapidly restores 'B' DNA because no its small ionic radius, it is ideally suited for specific inter-rearrangements of the strands are necessary. The require- action with nucleic acids. It is intriguing that many nucleases ment for an increased pH reflects the pK, of G (9.4) and and polymerases contain Zn2+ (Mildvan and Loeb 1979).
T (9.9) because the imino protons must be removed or be As well, Zn2+ is a good catalyst in the nonbiological eY- h ~-.g~ ~hle (Saenger 1984). As well, some of the sequence poly,i~alion of nucleic acids, especially since it favours effects on the rates of dismutation may be related to the the correct 3~-5~ orientation of the phospho~iester pK,s of the bases. For example, polyld(A-U)I and backbone (Bridson and Orgel 1980). Therefore, even though polyld(T C)]-poly[d(l-A)] both transform more rapidly a role for ~M~ DNA may appear to be unlikely in vivo 168 FIIOCHE!U. CELL BIOL VOL. 71, 1~93 b(eeause of the requirement for high pH and millimol~r Lee, J.S., Woodswonh, M.L., Latima, L.J.P., and Morgan, A.R.
Zn~t, its structure may ye~ provide clues to understanding 1984. Pyr-Pur DNAs containing 5-methylcytosine form stable the prebiotic chemistry of nucleic acids. triplexes at neutral pH. Nucleic Acids Res. 12: 6603-6613.
Lee. J.S., Burkholder, G.D., Latimet, L.J.P., Haug, 8.L., and Beese, L.S., and Steitz, T.A. 1991. Structural basis for the 3'-5' Braun, R.P 1987. A ~ ~' antibody to tripkx DNA binds ~Y- I ~ activity of E ~oli DNA f~l~.. aae l . a two metal to eukaryotlc .h.c,.. oso Nucleic Acids Res. 15: 1047-1061.
ion r-~~~ ' EM80 J. 10: 25-33 Lyamichev, V.l., Voloshin, O.N., Frank-tC: ~ ' '' M.D., and Bridson, P.K., and Orgel, L.E. 1980. Catalysis of accurate poly(C} Soyfer, V.N. 1991. Photofc~ty.' " a Of DNA triplexes. Nucleic directed synthesis of 3'-5' linked ~'i o -~ es by Znl~. Ac~ds Res. 19: 1633-1638.
J Mol. Biol. 144 567-577 '~ v Maher, L.l., Dervan, P.B., and Wold, B.l. 1990. Analysis of Chan, A., Kilkuskie, R., and Hanlon, S. 1979. Cc",elations promota-specificrepressionbyt.i~ " 'DNAcomplexesof between the duplex winding angle and the circular dichroism eukaryotic cell-free tIal~S~ . system. r - ~ - r, 29:
spectrum of calf thymus DNA. 13ic-' .1, 18: 84-91. 882~8826.
Cottoo, F., and Wilkinson, G. 1966. Advanced inorganic Marmur, 1., and Doty, P. 1962. D te. -' ~r of the base com-chemistry. 2nt ed. 1- -- :' , New York, London, Sydney. posltion of DNA from its thermal d - ation ~ f~ aIh.C.
D'Andrea, D.A., and Haseltine, W.A 1978 Sequence specific . Mo. io. 5: 1 - 18.
cleavage of DNA by the -- antibiotlcs nc ~ ~ - Mickel, S., Atena, V., and Bauer, W. 1977. Physical properties and bleomycin. Proc. Natl. Acad. Sci. U.S.A. 15. 3608-3612. and gel ele.l.ù,)l ~ ;, behaviour of R-12 derived plasmids.
Eichhorn G.L. 1962. Metal ions as stabili_~rs or d~t~ s of Nuclelc Acids Res. 4: 1465-1479.
DNA. Nature (Loodon), 194: 474-475 Mildvan, A.S., and Loeb, L.A. 1979. The role of metal ions in Eichhoro G.L., and Shin, Y.A. 1968. Ibteraction of metal ions the ~ - ' ' of DNA and RNA POIJ ~;. CRC Crit. Rev.
wnh~l~ andrelatedv~ L ' Xll l-Am-Chem- Mo8r~gCahnemARM~LeeB~~ll's6 F21~9~ jc414 Ik DE Murray NL and Evans, D.H., Lee, I.S., Morgan, A.R., and Olsen, R.K. 1982. Evans,D.E. 1979.Ethidiumbromidelluu., ,~ assays,Part 1.
uâing th~ A-T sp~cific ~ 1. iodn ~tfbjpolyld(A T)] synthesis Nucleic Acids Res 7 547-569.
1. 8iochem. 60: 131-136. Crit. Rev. Biochem. Mol. 8iol. 26: 151-226.
Guay, F., and E! . ' - ., A.L. 1979. Model ~ for the Rich, A., No-l" ' , A., and Wang, A.H.I. 1984. The chemistry ' - ~aion of silver I with pol~ '' - 1. Am. Chem. Soc. 101: and biology of left-handed DNA. Annu. Rev. 8iochem. 53:
626~6263 Hatopel, K.l, Crosson, P., and Lee, J S. 1991 Pol~ favor Rosenberg, 8, van Camp, L., Trosko, J.E., and Mansour, V.H.
DNA triplex formation at neutral pH. 8iochemistry 30 1969. Platinum cu~po- ' . a new class of potent ~ u 4455_4459 ' ' agents. Nature (London), 222: 385-386.
Haner, R., and Dervan, P.8. 1990. Single-strand DNA triple-helix Sa nger, W. 1984. Principles of nuckic acid structure. Springer-formation. P:c-'- ' - ~ 29. 9761-9765 erag, ew or .
Haniford, D.8., and P~ , D.E. 1983. Facil~ transition of Scaria, P-Vj, and Shaf~r, R.H. 1991. 8inding of ethidium to a poly[d(TG)] poly[d(CA)I into a left handed helix in ph~,iolo~J DNA tnp e e tx. . 8iol. Chem. 266: 5417-5423.
''-'- Nature (London), 302. 632-634 Sklenar, V., and Feigon, 1. 1990. Formation of a stabk tripla~
Hodgson, D J. 1977. The ste.. ' '- ~ of metal complexes of from a single DNA strand. Nature (London), 34S: 836-838.
nucleic acid ~ - . Prog. Inorg Chem 23- 211 -254 S~ ' ' ~ n, V, and S ' ' _' M. 1979. The crystal struc-Hore, P.l. 1983. 8inomial composite pulse sequences for water tures of nuclelc acids and their CO~ CRC Crit. Rev.
suyy.c ' ~ in NMR speara. I. Magn. Reson. 54. 539-542 8iochem. Mol. 8iol. 6: 245-336.
Htun, H and Dahlberg, I.EM988s. Single strands triple strands Timsit, Y, Vilbois, E., and Moras, D. 1991. 8as~ pairing shift and kinks in H-DNA. Science (Washington D.C.), 241: m the ma30r groove of (CA)1 traas by 8-DNA crystal struc-1791-1796 tures. ature ( on on), 354: 167-1 0.
Kohwi, Y., and Kohwi-S~ '_ . T 1988 M~ ion- Yagil, G- 1991. Paranemic structures of DNA and their role in dependent triple-hclix struaure formed by pyr-pur xquences DNA unwinding. CRC Crit. Rev. 8iochem. Mol. 8iol. 26:
in supercoiled DNA. Proc. Natl. Acad. Sci. 85: 3781-3785. ' . .
Latham~ I A, and Cech, t R. 1989. Defining the insid~ and out- Zahler, A.M., Wllllamson, J.R., C~ch, T.R., and Pr~scott, D.M.
side of a cata3ytic RNA molecule. Science (Washington, D.C.) 1991. Inhlbition of tel~. . ~e by G~uartet DNA structures.
245: 276-282. Nature (London), 350: 718-720.
Lee, J.S. 1990. Thc stability of polypurine tetraplexes in the Zimmer, C., and Luck, G. 1974. Conformation and reaaivity of pre~ence of mono and divalent ions. Nucleic Acids Res. 18: DNA Clrcular dichroism studies, Vl. 8iochem. 8iophys. Acta, 6057-6060.

M-DNA

Plea~e ~ind the ~ ched flgure demon~tra~ng the nudease re~i6tance of M-DNA. Ihe ~mount of duplex DNA remalnlng ~~ a func~on of ~me ~a~
~6~et by ~e e~idium fluor~ ,ce a~say (under cont~tion~ where M-DN~
rapidl~r reve~s to 8-DNA so ~t e~h~ can blnd). The d~ on wa~
p~,fo...~ed at 37 ~C ln 10 mM Tns-Hcl pH 7.4, 5 mM MgC12, lmM N~C12, 1mg/ml g~ , and 0.2 ~g/~l DNase I. Ihe M-l)~ s~mple wa~ prefarmet ~t pH 9 bef~ adding ~o tht dige~on buffer whereas ~e ~DNA W,~18 ~dtled ~rectly. ~he M-ON~ 18 completely res~ while ~e ~DNA ~9 di8e~ted ln ~bout 10 m~n. Note ~at ~i~ expedment ~ hows th~t ~e Ni fonn of M-DNA is ~te st~le under ph~iologlcal condl~ which retate~ to ih utility in DNA lmmw~ sense technologle~.

.
o~
C~ 1.2 ~ DN~

t 0.
~0.0 ~0.~
0.2 -O

~ (~)

Claims

Claims I claim:

(1) A DNA molecule comprising a metal-containing DNA
molecule, an electron donor electrically coupled to a first end of the metal-containing DNA duplex and an electron-acceptor electrically coupled to a second end of the metal-containing DNA duplex, the metal-containing DNA
duplex comprising a divalent metal cation substituted for the imino protons of a DNA base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+, Co2+, and Ni2+.

(2) The use of a metal-containing DNA duplex to carry electrons from a first end of a DNA duplex to a second end of the DNA duplex, the metal-containing DNA duplex comprising a divalent metal cation substituted for the imino protons of a DNA base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+, Co2+, and Ni2+.

(3) The use of metal-containing DNA for DNA immunization the metal-containing DNA duplex comprising a divalent metal cation substituted for the imino protons of a DNA
base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+, Co2+, and Ni2+.

(4) The method of immunizing a host comprising injecting the host with metal-containing DNA, the metal-containing DNA duplex comprising a divalent metal cation substituted for the imino protons of a DNA base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+ Co2+ and Ni2+

(5) The use of metal-containing DNA to inhibit expression of a complementary gene sequence, the metal-containing DNA duplex comprising a divalent metal cation substituted for the imino protons of a DNA base selected from the group consisting of thymine and guanine, the divalent metal cation being selected from the group consisting of Zn2+, Co2+, and Ni2+

(6) A method of detecting a complementary DNA sequence comprising:
providing a first DNA sequence;
providing a second complementary DNA sequence under conditions which allow the first and second DNA sequences to form a metal-containing DNA duplex wherein a divalent metal cation is substituted for the imino protons of thymine or guanine base pairs;
detecting the formation of the metal-containing DNA
duplex.
CA002218443A 1997-12-16 1997-12-16 Metal-containing dna Abandoned CA2218443A1 (en)

Priority Applications (12)

Application Number Priority Date Filing Date Title
CA002218443A CA2218443A1 (en) 1997-12-16 1997-12-16 Metal-containing dna
CA002229386A CA2229386A1 (en) 1997-12-16 1998-02-11 Conductive metal-containing nucleic acids
CA002255113A CA2255113C (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
ES98960961T ES2210847T3 (en) 1997-12-16 1998-12-16 NUCLEIC ACIDS CONTAINING DRIVING METALS.
EP98960961A EP1042344B1 (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
PCT/CA1998/001149 WO1999031115A1 (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
DE69819780T DE69819780T2 (en) 1997-12-16 1998-12-16 CONDUCTIVE METAL CONTAINING NUCLEIC ACIDS
AT98960961T ATE254134T1 (en) 1997-12-16 1998-12-16 CONDUCTIVE METAL-CONTAINING NUCLEIC ACIDS
AU16562/99A AU1656299A (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
US09/213,052 US6432641B1 (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
JP2000539038A JP2002508386A (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids
EP03023704A EP1382610A3 (en) 1997-12-16 1998-12-16 Conductive metal-containing nucleic acids

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CA002218443A CA2218443A1 (en) 1997-12-16 1997-12-16 Metal-containing dna

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