Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.

(3′-2′)-R-L-Threose nucleic acid (TNA, Figure 1A) is an unnatural nucleic acid that was identified during an extensive evaluation of alternative sugar-phosphate backbones aimed at explaining the structure of the biological nucleic acids. 1,2 TNA possesses the ability to specifically base-pair with RNA, DNA, and itself. 2 This capability, together with the chemical simplicity of threose relative to ribose, suggests that TNA could have acted as an evolutionary competitor of RNA or even have preceded RNA as the genetic molecule of life. We are attempting to investigate the functional potential of TNA by implementing an in vitro selection scheme for TNA. 3,4 Here, we show that a mutant archaeal family B DNA polymerase is capable of polymerizing more than 50 nucleotides of TNA on a DNA template. We also demonstrate the display of single-stranded TNA covalently linked to its encoding duplex DNA, thus enabling the selection of functional TNA sequences and the amplification or recovery of the attached DNA.We and others have previously shown that certain family B archaeal DNA polymerases possess the ability to synthesize limited stretches of TNA on a DNA template. 5,6 Our recent synthesis of all four TNA triphosphates (tNTPs) enabled us to test polymerases for more extensive activity. 7 The Therminator DNA polymerase is an engineered exonuclease-deficient form of "9°N" DNA polymerase containing an A485L mutation. 8 It is capable of efficiently incorporating a wide spectrum of modified nucleotides. We tested the ability of this polymerase to accept tNTPs as substrates using a DNA primer/template construct containing a 50-nucleotide singlestranded template region in which all four DNA nucleobases were represented ( Figure 1B). Since previous work had shown that pairing diaminopurine opposite thymine increases the efficiencies of both template-directed ligation and polymerization, 5,9 we used diaminopurine triphosphate (tDTP) instead of tATP. The Therminator polymerase catalyzed the synthesis of >20% full-length 50-nucleotide TNA product within 24 h ( Figure 1C).We reasoned that if transcribed TNA could be covalently linked to its DNA template, we could perform functional selections for TNA molecules and rescue the successful genotypes by PCR amplification of the attached DNA. This approach is analogous to the selection of functional peptides and proteins by mRNA display; 10 the use of DNA display for peptide and PNA selections has also been proposed. 10,11 By starting with a library of single-stranded DNA hairpins, the 3′ end of each hairpin could act as a primer for TNA transcription across the randomized DNA template region (Figure 2A). A primer annealed to the loop region of the hairpin could then initiate strand-displacement synthesis, liberating the TNA strand to allow folding and linearizing the DNA template by making it double-stranded.To test this idea, we synthesized a single-stranded DNA hairpin and transcribed the 60-nucleotide single-stranded region using the Therminator DNA polymerase and tNTPs ( Figure 2B). N...