Synthesis and properties of size-expanded DNAs: toward designed, functional genetic systems

Abstract

We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA ("expanded DNA") and yDNA ("wide DNA"). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids.

Figure 1

Examples of natural and nonnatural pur-pyr pairs: A-T, Gs-Th, isoG-isoC. All of these conform to Watson and Crick’s purine-pyrimidine structural paradigm for the DNA double helix.

Figure 2

The four base pairing schemes of xDNA. xDNA is comprised of eight bases, while natural DNA has four bases with two pairing schemes.

Figure 3

Illustration of nucleobase size differences that lead to two classes of expanded DNA helices. (A) Watson-Crick DNA strands are complementary in size. (B) An all-expanded xDNA strand is size complementary to natural DNA. (C) Mixing expanded and non-expanded bases on a strand (8-base xDNA) results in a size mismatch (orthogonality) with natural DNA. (D) 8-base xDNA can be complementary to another 8-base xDNA strand.

Figure 4

Fluorescence spectra of two xDNA nucleosides (dxC and dxG) in methanol., Emission spectra are shown with solid lines, and excitation spectra with dashed lines. All xDNA and yDNA nucleobases synthesized to date are efficient fluorophores.

Figure 5

High stacking proficiency of xDNA and yDNA bases.,,, Free energies for stacking (kcal/mol, 37 °C) were measured at the end of a short DNA duplex. The strong stacking of the expanded bases is likely due both to stronger van der Waals forces and the hydrophobic effect.

Figure 6

Thermal denaturation curves showing the high stability of an xDNA helix compared to DNA of the same sequence.

Figure 7

Structures of xDNA measured by NMR, with comparison to DNA (right). (A) Space-filling models showing wider grooves of xDNA, but similar right-handed helical turn. (B) View of three adjacent xDNA pairs, showing large surface area of overlap compared to DNA.

Figure 8

Graphs showing base pairing selectivity of xDNA (A) compared to DNA (B), as measured by melting temperatures of short duplexes containing a single mismatched pair as shown. (Reprinted with permission of Angew. Chem. Int. Ed.)

Figure 9

Preliminary data showing the ability of DNA polymerase I (Klenow fragment) to function with the xA base. (A) Polyacrylamide gel showing addition of dxA to the end of a primer opposite A, T, G, or C; (B) histogram showing relative efficiency of insertion of dTTP opposite xA. Note log scale of efficiency; natural TA base pair efficiency is given for comparison at right.

Figure 10

Pairing scheme of doublewide DNA (yyDNA). The proposed double helix has an expected diameter nearly 5Å wider than natural DNA. (A) Expected base pair. structures; (B) comparison of DNA pair size and geometry with yyDNA pair.

Scheme 1

Synthetic route for preparation of dxA phosphoramidite derivative. Conditions: (a) KMnO₄, tBuOH, H₂O, 70 °C, 1.5 h, 60%; (b) Ac₂O 155 °C, 3 h, 90%; (c) TMSN₃, 90–95 °C, 3 h; (d) formamidine acetate, DMF, 155 °C, 3hr, 55% (two steps); (e) P₂S₅, Py, 140 °C, 36 h, 100%; (f) CH₃I, KOH, 1 h, 64%; (g) NaH, CH₃CN, Hoffer’s chlorosugar, 6 h,.37%; (h) NH₃, EtOH, 140 °C, 36 h, 61%; (i) N,N-dimethylacetamide dimethylacetal, MeOH, 80 °C, 72 h, 69%; (j) DMTrCl, DIPEA, Py, 23 °C 6 h, 54%; (k) N, N-diisopropyl-cyanoethyl-chlorophosphoramidite, CH₂Cl₂, DIPEA, 23 °C, 5 h, 90%. (Reprinted with permission of J. Am. Chem. Soc.)

Scheme 2

Synthetic route for preparation of dxT phosphoramidite derivative. Conditions: a). ICl, HCl(aq.), 96%; b) urea, 150 °C, 92%; c) POCl₃, N, N-diethylaniline, reflux, 90%; d) NaOCH₃/CH₃OH, reflux, 87%; e) 1,2-dehydro-3-O-(t-butyldiphenylsilyl)-5-hydroxymethyl-furan, Pd(OAc) ₂, AsPh₃, N(Bu) ₃, 75 °C, 64%; f) TBAF, THF, 0 °C, 84%; g) NaB(OAc)₃H, THF, AcOH, 15 °C, 93%; h) NaI, AcOH, 60 °C, 100%; i) 4,4′-dimethoxytrityl chloride, DIPEA, pyridine, 84%; j) N,N-diisopropylammonium tetrazolide, 2-cyanoethyl tetraisopropylphosphoramidite, CH₂Cl₂, 82%. (Reprinted with permission of J. Am. Chem, Soc.)