Recently an amino-functionalized deoxyuridine was used to select for a dna-based atp aptamer ( ). By comparison, no mutations were observed for the sequences obtained using utp. The majority of these studies have focused on the modification and selection of dna, presumably due to its enhanced chemical stability relative to rna, but rna plays a more active role in the cell and might be expected to exhibit a greater catalytic potential than dna.
Additional templates were designed and used that contained six sites coding for uridine residues with no coded uridine residues in the reverse transcription primer binding site. Rnas that each contained 15 (25) unh b shows the digestion analysis of products from an rna 15mer containing only three ush residues. Since altmans and cechs initial discovery and the observation that some rnas exhibit enhanced cleavage activity ( ), several natural rnas with catalytic motifs have been also been identified ( ) and numerous rna catalytic motifs and aptamers have been selected andor evolved through selection or selex procedures involve the isolation and amplification of rna and dna sequences, which exhibit selected properties, from a large pool of random sequences through repeated cycles.
The polymerization reaction to generate the rna 5mer contained 40 mm trishcl (ph 8. Rna 15mer transcript 5-ggcguacguacgua-3 in which the u residues are ush. The solution was hydrogenated in a parr hydrogenation apparatus at 85 p.
Transcription of rna pools with t7 rna polymerase and unh or ush occurred with efficiencies of 43 and 29, respectively, of the values obtained for native utp transcription. High fidelity incorporation of the analogs is observed, but owing to possible secondary structure effects, some deletions are apparent when using the ush derivative. Such artifacts have been reported previously in both reverse transcription ( ).
Pcr was performed in a ptc-200 peltier thermocycler (mj research, waltham, ma), with a typical cycle of 94c for 2 min, 92c for 2 min, 55c for 1 min and 72c for 1 min and cycled between 92 and 55c (see also cloning protocol). Additionally, the thiol is an effective ligand for metal coordination. Beckman 114m solvent delivery units were used to obtain a flow rate of 7 mlmin with a gradient of 01 m nacl aqueous solution buffered with 20 mm trishcl (ph 7.
Cloning and sequencing of the amplified dsdna oligos obtained from the unh -containing rna revealed that transcription and reverse transcription had proceeded with high fidelity. An additional explanation for reduced activity with ush is the relatively high concentration of dtt present in those transcription assays. Under those conditions, transcription efficiency increased to a value of 10 of that observed with utp (data not shown). The protonated amine, as a lysine-like functionality, could be valuable for its ability to interact with the charged phosphate backbone of nucleic acid aptamers and facilitate folding of the rna into new types of active complexes. Additionally, helene and co-workers have simultaneously incorporated two modified deoxynucleotides into a dna transcript ( ).
Uv analyses were performed on a beckman du 640 spectrophotometer. Transcripts for use in reverse transcription reactions and analysis by snake venom phosphodiesterasereverse phase hplc were isolated from the gels by passive elution in water and precipitated using 300 mm nacl and 3 vol absolute ethanol. Molecular structure descriptors in the computer-aided design of biologically active compounds h. Triton x-100, 5 mm each nucleoside triphosphates, 510 ci - pctp, 200 nm dsdna template and 400 ul t7 rna polymerase. T7 rna polymerase (200 ul) was purchased from ambion (austin, tx) or prepared from over-producing plasmid par1219 according to the method of zawadzki and gross ( reverse transcriptase (200 ul) was purchased from life technologies (grand island, ny).
The reverse transcription reactions with template containing these analog residues are also effective, provided that their presence in the primer binding site is minimized. Kcl, 6 mm mgcl , 10 mm dtt, 0. Both transcripts exhibited a gel shift effect during page due to reduced mobility relative to that of the native transcript (fig. Ush is deprotected immediately before transcription using 10 mm dtt and then used directly without purification. When unh was used in the transcription reaction to produce a pool of rna 100mers, containing a randomized tract of 60 nt with 25 of the transcript sequence as unh (of which six were contained in the reverse transcription primer binding site), transcription efficiency was 43 relative to that with utp.
The efficiency of reverse transcription in this case for the unh -containing transcript was observed to be indistinguishable from that of the native sequence (fig. A 100mer template was transcribed containing a randomized tract of 60 nt, where the transcript contained 25 uridines in the random region and six uridines in the reverse transcription primer binding site. Propargylamine was protected with trifluoroacetic anhydride, coupled to 5-iodouridine using previously described procedures ( ) and reduced by hydrogenation to the n-protected 5-(3-aminopropyl)uridine derivative. This research was supported by the nsf (mcb 0077667). Their kinetic, potential and total energies were also calculated through md simulation. Nmr spectra were obtained on 400 and 500 mhz varian ft-nmr spectrometers using the deuterated solvent as an internal standard. The 60mer transcript contained six uridines but no uridines in the reverse transcription primer binding site. Transcription was also examined in the presence of reduced concentrations of atp, ctp and gtp (at 2. P nmr spectra were obtained on a 500 mhz varian ft-nmr using phosphoric acid as an external standard. Anion exchange chromatography was performed using a 250 ml glass column packed with sephadex deae-a25 from pharmacia (piscataway, nj).Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for ...