Functional isoDNA aptamers: modified thrombin binding aptamers with a 2'-5'-linked sugar-phosphate backbone (isoTBA).

The regioisomeric 3'-deoxy-2'-5'-linked thrombin binding DNA aptamers (isoTBAs) were chemically synthesized and their ability to form unimolecular anti-parallel G-quadruplexes in the presence of K(+) ions was evaluated. These modified sequences retain the function of the native thrombin binding aptamer (TBA), exhibit better stability against exonuclease and are capable of slowing down the process of blood clotting.

Detection and knockdown of microRNA-34a using thioacetamido nucleic acid.

Thioacetamido nucleic acids (TANA) contain a backbone modification of dinucleotides consisting of a 5-atom amide linker N3'-COCH2-S-CH2 at thymidine or thymidine-cytidine dimer blocks. Here, the chemical synthesis of the TANA linked 5-methyl-cytidine-cytidine ((Me)cc) dimer block and its incorporation into the DNA sequence, complementary to human microRNA 34 (miR-34) is described. Further, for the first time, we demonstrate the biological applications of TANA modified oligonucleotides in detection and intracellular knockdown of a cancer related microRNA in comparison with DNA containing locked nucleic acid (LNA) and 2'-O-methyl modifications. The human microRNA miR-34 is a pro-apoptotic microRNA under the transcriptional control of protein 53 (p53). It gets expressed in response to DNA damage and regulates several cell cycle and apoptosis related targets. Here, we show that the TANA modified antisense oligonucleotide binds specifically to miR-34a, allowing its detection using primer extension. We also show that, using the TANA modified antisense oligonucleotide against miR-34a, intracellular levels of miR-34 can be reduced, and consequently, the expression of its target oncogene V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN) is enhanced. Further, we assessed the toxicity and serum stability of the oligonucleotide to conclude that it is suitable for detection and modulation of the vital biomarker and tumor suppressor microRNA.

Synthesis and structural studies of S-type/N-type-locked/frozen nucleoside analogues and their incorporation in RNA-selective, nuclease resistant 2'-5' linked oligonucleotides.

2'-endo locked or frozen (S-type)/3'-endo locked or frozen (N-type) nucleoside analogues were synthesized. Conformational analysis based on (3)J(HH) and NOE measurements is presented which is further confirmed by X-ray crystal structural studies. 2'-5'isoDNA oligonucleotides (ON) were synthesized using these modified nucleoside analogues and UV-T(m) studies of the resultant 2'-5'isoDNA : RNA duplexes reflect the site- and sequence-dependent effects and confirm that the S-type sugar conformations were preferred over the N-type sugar geometry in such duplexes.

Probing the furanose conformation in the 2'-5'strand of isoDNA : RNA duplexes by freezing the nucleoside conformations.

Sugar conformations in the isoDNA strand of isoDNA : RNA duplexes are preferred S-type locked/frozen in contrast to N-type locked conformations preferred in DNA : RNA duplexes.

Effects for the incorporation of five-atom thioacetamido nucleic acid (TANA) backbone on hybridization thermodynamics and kinetics of DNA duplexes.

Thermodynamic studies of nucleic acids serve not only to widen our understanding on the nature and strength of forces that stabilize nucleic acids in a myriad of structural states they assume but also to facilitate the development of databases that could be used to predict the stability and selectivity of probe/primer-sets required in a broad range of nucleic acid-based diagnostic and therapeutic protocols. In the current study, we investigated the effect of a novel, backbone-modified "thioacetamido linkage" (TANA) on thermodynamics of hybridization, binding kinetics, and conformation of a DNA duplex. The modification comprises of an extended five-atom amide (N3'-CO-CH2-S-CH2) linker, as opposed to four-atom phosphodiester linker backbone present in DNA. One to three TANA-substitutions have been introduced in the linker backbone of two thymidine residues of one of the strand of the DNA duplex. Using spectroscopic and calorimetric techniques, we observed that TANA destabilizes the DNA helix by lowering the favorable enthalpy parameter of duplex formation. TANA x DNA duplexes were found to adopt a conformation toward an A-type duplex as shown by circular dichroism spectroscopy studies. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, deltaCp, accompanying the duplex formation. The average deltaCp, change per duplex was found to be 832.5 cal mol(-1) K(-1), giving an average base-pair change of 59.5 cal (mol of base pairs)(-1) K(-1). Hybridization kinetic measurements using surface plasmon resonance indicated a decrease in binding affinity parameter (KA) that originates from higher dissociation rate constants (k(d)). Furthermore, optical melting studies showed that increasing the number of modifications results in a modest change in the number of counterions taken up during duplex formation.

Synthesis of locked S-type and locked N-type uridine monomer units for incorporation in 2'-5' RNA:3'-5'RNA duplexes.

To delineate the structural requirements of 2'-5' RNA:3'-5' RNA duplexes, we report the synthesis of N-type locked, S-type locked and 3'-fluoro-2'-phosphoramidite building blocks. The consequent incorporation of these three novel monomers into 2'-5' linked oligomers and their biophysical implications on the stability of the said duplexes will have explicit importance towards development into therapeutic oligomers. The intrinsic stability of 2'-5' phosphodiester linkage as opposed to 3'-5' linked oligomers will be an added advantage.

Synthesis and RNA binding selectivity of oligonucleotides modified with five-atom thioacetamido nucleic acid backbone structures.

Convenient chemical synthesis and incorporation of dithymidine and thymidine-cytidine dimer blocks connected with a five-atom amide linker N3'-CO-CH2-S-CH2 into oligonucleotides (ONs) are reported. The UV-Tm experiments for binding affinities of these mixed backbone ONs with complementary DNA and RNA sequences revealed important results such as significantly higher RNA-binding selectivity as compared with complementary DNA. NMR studies of the dimer blocks suggested a marginal increase in the N-type sugar conformations over that of the native DNA.

Sugar-thioacetamide backbone in oligodeoxyribonucleosides for specific recognition of nucleic acids.

The amide linkage being shorter than the natural phosphate linkage, an additional atom is introduced into oligodeoxyribonucleosides (ODNs) with sugar-thioacetamide backbone that show very good RNA recognition properties.