Wang Xiaoyu, Hoshika Shuichi, Peterson Raymond J, Kim Myong-Jung, Benner Steven A, Kahn Jason D
Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States.
Foundation for Applied Molecular Evolution , 13709 Progress Boulevard, No. 7, Alachua, Florida 32615, United States.
ACS Synth Biol. 2017 May 19;6(5):782-792. doi: 10.1021/acssynbio.6b00224. Epub 2017 Feb 9.
Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°) for oligonucleotide hybridization were extracted. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair observed for C:O-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodynamics of oligonucleotides containing Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.
呈现氢键供体和受体基团非沃森-克里克排列方式的合成核碱基能够形成额外的核苷酸对，这些核苷酸对可稳定双链DNA，且不依赖于标准的A:T和G:C对。2-氨基-3-硝基吡啶-6-酮2'-脱氧核糖核苷（在小分子的沃森-克里克面上呈现{供体-供体-受体}氢键模式，简称为Z）与咪唑并[1,2-a]-1,3,5-三嗪-4(8H)酮2'-脱氧核糖核苷（在大分子上呈现{受体-受体-供体}氢键模式，简称为P）之间的碱基对就是这些额外碱基对之一，针对该碱基对已经开展了大量的分子生物学研究。在此，我们报告紫外吸收熔解测量结果，并确定含有Z和P的DNA链结合形成包含Z:P对以及各种由Z和P组成的错配双链体的能量学。所有测量均在1 M NaCl的缓冲液（10 mM 二甲胂酸钠，0.5 mM 乙二胺四乙酸，pH 7.0）中进行。提取了寡核苷酸杂交的热力学参数（ΔH°、ΔS°和ΔG°）。与考虑几何和氢键互补性的沃森-克里克模型一致，发现Z:P对比任何涉及非标准核苷酸的错配更有助于双链体稳定性。此外，Z:P对比C:G对更稳定。发现Z:G对是最稳定的错配，形成去质子化的错配碱基对或类似于稳定的T:G错配的摆动碱基对。C:P对稳定性较差，可能类似于C:O-甲基-G中观察到的摆动对，其中嘧啶被位移到小沟中。Z:A和T:P错配稳定性要低得多。提供了预测含有Z和P碱基的寡核苷酸热力学的参数。这代表了对合成遗传系统进行此类研究的首例。
