All PDF files with the numbers refer to paper numbers in the original list of publications.
(A) We have recently reported [PDF 337,
our studies on the conjugation of photoreactive Ru2+ complex
to oligonucleotides (ODNs), which give stable duplex with the complementary
target DNA strand. These functionalized DNA duplexes bearing photoreactive
Ru2+complex can be specifically photolyzed to give the
reactive aqua derivative, [Ru(tpy)(dppz)(H2O)]2+-ODN,in situ,
which successfully cross-links to give photoproduct(s) with the target
DNA strand in a sequence specific manner. Thus, the stable precursor of
aqua-ruthenium complex, monofunctional polypyridyl ruthenium complex:
(tpy = 2,2':6',2''-terpyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine),
[Ru(tpy)(dppz)(CH3CN)]2+ has been site-specifically tethered to ODN, for the first time, both by solid phase synthesis and post-synthetic modifications.
(i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH3CN)]2+-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl]+ complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl ligand by acetonitrile in the coordination sphere of the Ru2+-label.
(ii) In the second approach, the single modified (3'-, or 5'- or middle-modified-) or 3',5'-bis-modified Ru2+-ODN conjugates were prepared in ca 50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino-linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI).
[Ru(tpy)(dppz)(CH3CN)]2+-ODNs form highly stabilized ODN·DNA duplexes compared to the unlabeled counterpart ( Tm varies from 8.4 to 23.6°C) as a result of intercalation of the dppz moiety; they undergo clean and selective photodissociation of CH3CN ligand to give the corresponding aqua complex, [Ru(tpy)(dppz)(H2O)]2+-ODNs (in the aqueous medium), which is evidenced from the change of their UV-vis absorption properties and the detection of the naked Ru2+-ODN ions generated in the course of the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis.
Thus, when [Ru(tpy)(dppz)(CH3CN)]2+-ODN conjugate was hybridized to the complementary guanine (G)-rich target strand (T), and photolyzed in a buffer (pH 6.8), the corresponding aqua complex formed in situ immediately reacted with the G residue of the opposite strand giving the cross-linked product. The highest yield (34%) of the photo cross-linked product obtained was with the ODN carrying two reactive Ru2+ centers at both 3'- and 5'-ends. For ODNs carrying only one Ru2+ complex, the yield of the cross-linked adduct in the corresponding duplex is found to decrease in the following order: 3'-Ru2+ ODN (22%) > 5'-Ru2+ ODN (9%) > middle-Ru2+ ODN (7%). It was also found that the photo cross-coupling efficiency of the tethered Ru2+ complex with the target T strand decreased as the stabilization of the resulting duplex increased: 3'-Ru2+ ODN (VIII·T) ( Tmb = 7°C) < 5'-Ru2+ ODN (V·T) ( Tmb = 16°C) < middle-Ru2+ ODN (VII·T) ( Tmb = 24.3°C, Table 1). This shows that with the rigidly packed structure, as in the duplex with middle-Ru2+ ODN, the metal center flexibility is considerably reduced, and consequently the accessibility of target G residue by the aquaruthenium moiety becomes severely restricted, which results in a poor yield in the cross-coupling reaction. The cross-linked product was characterized by PAGE, followed by MALDI-TOF MS.
(B) Since RNA cleavage site has the 2'-endo geometry in the self-cleaving lariat-RNAs, our studies have given the first indications that the nucleolytic DNA has to form a bulge or a hairpin in order to efficiently cleave the target RNA because the nucleotides in those bulge or hairpin will have also 2'-endo sugar geometry (as also found by Tinoco et al in their RNA hairpin studies) which is necessary for bringing the 2'-OH in the close proximity of the vicinal phosphate. At least one pair of mismatched mononucleotide residues should make up the mismatched bulge in the duplex and create the necessary conformational change of the sugar phosphate backbone within the bulge, similar to the ones found in self-cleaving lariat RNAs. In order to further promote hydrolytic cleavage of the target RNA within the bulge one or more tethered functional groups need to be incorporated into the bulge by attaching them to the nucleolytic DNA strand. These functional groups can be similar to those found in the active-site cavity of the RNase A enzyme, but can also consist of metal chelating ligands, which can direct the metal ion (i.e. Cu3+, Fe3+, Ru3+, Os3+, Eu3+, Tb3+, Sm3+) to a certain position within the target RNA strand and thereby induce RNA cleavage with a catalytic turnover.
Since the nucleolytic DNA should be reasonably short (12-14-mer) and mismatches act destabilizing, a 'stacker' at either or both the 3'- and 5'-ends of the DNA is being used to enchance duplex stability. This has been shown to enhance the resistance to cellular exonucleases of the antisense or antigene oligonucleotides and thereby increase their stability and therapeutic efficiency.
In this regard we are engineering the antisense strands, which upon hybridization locally change the conformation of the sugar-phosphate backbone of the target RNA strand to be similar to self-cleaving lariat RNAs, and will orchestrate the catalytic self-cleavage.