Optimization of an unnatural base pair toward natural-like replication.

Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivatives are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM:d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the minimum overall fidelity ranges from 10(3) to 10(4). In addition, the data allow us to propose a generalized model of unnatural base pair replication, which should aid in further optimization of the unnatural base pair and possibly in the design of additional unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.

[1]  T. Spratt.  Identification of hydrogen bonds between Escherichia coli DNA polymerase I (Klenow fragment) and the minor groove of DNA by amino acid substitution of the polymerase and atomic substitution of the DNA. , 2001, Biochemistry.

[2]  P. Schultz,et al.  Enzymatic phosphorylation of unnatural nucleosides. , 2002, Journal of the American Chemical Society.

[3]  J. SantaLucia,et al.  Thermodynamic parameters for DNA sequences with dangling ends. , 2000, Nucleic acids research.

[4]  Yoko Harada,et al.  An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA , 2006, Nature Methods.

[5]  David Norwood,et al.  Multiplexed detection of anthrax-related toxin genes. , 2006, The Journal of molecular diagnostics : JMD.

[6]  Steven A. Benner,et al.  Enzymatic incorporation of a new base pair into DNA and RNA , 1989 .

[7]  Aaron M. Leconte,et al.  Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. , 2008, Journal of the American Chemical Society.

[8]  F. Romesberg,et al.  Efforts to expand the genetic alphabet: identification of a replicable unnatural DNA self-pair. , 2004, Journal of the American Chemical Society.

[9]  F. Romesberg,et al.  Substituent effects on the pairing and polymerase recognition of simple unnatural base pairs , 2006, Nucleic acids research.

[10]  J. Prudent.  Using expanded genetic alphabets to simplify high-throughput genetic testing , 2006, Expert review of molecular diagnostics.

[11]  F. Romesberg,et al.  Rational Design of an Unnatural Base Pair with Increased Kinetic Selectivity , 2000 .

[12]  V. Danilov.  On the nature of stability of the nucleotide base associates in water solution , 1975, Molecular Biology Reports.

[13]  Steven A. Benner,et al.  Enzymatic incorporation of a third nucleobase pair , 2007, Nucleic acids research.

[14]  Aaron M. Leconte,et al.  Stability and polymerase recognition of pyridine nucleobase analogues: role of minor-groove H-bond acceptors. , 2006, Angewandte Chemie.

[15]  Anthony D. Keefe,et al.  SELEX with modified nucleotides. , 2008, Current opinion in chemical biology.

[16]  T. Spratt,et al.  Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-bonding Fork from Arg668 to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication*[boxs] , 2004, Journal of Biological Chemistry.

[17]  S. Yokoyama,et al.  An efficient unnatural base pair for a base-pair-expanded transcription system. , 2005, Journal of the American Chemical Society.

[18]  Aaron M. Leconte,et al.  Minor groove hydrogen bonds and the replication of unnatural base pairs. , 2007, Journal of the American Chemical Society.

[19]  Aaron M. Leconte,et al.  Polymerase evolution: efforts toward expansion of the genetic code. , 2005, Journal of the American Chemical Society.

[20]  Eric T. Kool,et al.  A specific partner for abasic damage in DNA , 1999, Nature.

[21]  T. Ellis,et al.  Multiplex Genotyping of Human Minor Histocompatibility Antigens , 2005 .

[22]  F. Romesberg,et al.  Optimization of unnatural base pair packing for polymerase recognition. , 2006, Journal of the American Chemical Society.

[23]  E. Kool,et al.  Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases. , 2000, Biochemistry.

[24]  Peter G Schultz,et al.  Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs. , 2007, Journal of the American Chemical Society.

[25]  Floyd E. Romesberg,et al.  Efforts toward Expansion of the Genetic Alphabet: DNA Polymerase Recognition of a Highly Stable, Self-Pairing Hydrophobic Base , 1999 .

[26]  Nadrian C. Seeman,et al.  An Overview of Structural DNA Nanotechnology , 2007, Molecular biotechnology.

[27]  Irene Lee,et al.  Hydrophobicity, shape, and pi-electron contributions during translesion DNA synthesis. , 2006, Journal of the American Chemical Society.

[28]  G. Waksman,et al.  Crystal structures of a ddATP‐, ddTTP‐, ddCTP, and ddGTP‐ trapped ternary complex of Klentaq1: Insights into nucleotide incorporation and selectivity , 2001, Protein science : a publication of the Protein Society.

[29]  S. Yokoyama,et al.  An efficient unnatural base pair for PCR amplification. , 2007, Journal of the American Chemical Society.

[30]  Aaron M. Leconte,et al.  Polymerase Recognition and Stability of Fluoro‐Substituted Pyridone Nucleobase Analogues , 2007, Chembiochem : a European journal of chemical biology.

[31]  Steven A Benner,et al.  Nucleobase pairing in expanded Watson-Crick-like genetic information systems. , 2003, Structure.

[32]  E. Kool,et al.  Selective and Stable DNA Base Pairing without Hydrogen Bonds. , 1998, Journal of the American Chemical Society.

[33]  J. M. Healy,et al.  Complex target SELEX. , 2008, Accounts of chemical research.

[34]  N. Seeman.  From genes to machines: DNA nanomechanical devices. , 2005, Trends in biochemical sciences.

[35]  Ichiro Hirao,et al.  Unnatural base pair systems for DNA/RNA-based biotechnology. , 2006, Current opinion in chemical biology.

[36]  Aaron M. Leconte,et al.  Efforts towards expansion of the genetic alphabet: pyridone and methyl pyridone nucleobases. , 2006, Angewandte Chemie.

[37]  M. Moser,et al.  MultiCode-RTx Real-Time PCR System for Detection of Subpopulations of K65R Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant Viruses in Clinical Samples , 2006, Journal of Clinical Microbiology.

[38]  Eric B. Roesch,et al.  Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet , 2004, Clinical chemistry.

[39]  Eric B. Roesch,et al.  Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system. , 2003, Clinical chemistry.

[40]  S. Creighton,et al.  Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. , 1995, Methods in enzymology.

[41]  P. Schultz,et al.  The effect of minor-groove hydrogen-bond acceptors and donors on the stability and replication of four unnatural base pairs. , 2003, Journal of the American Chemical Society.

[42]  F. Romesberg,et al.  Determinants of unnatural nucleobase stability and polymerase recognition. , 2003, Journal of the American Chemical Society.

[43]  M. Ieven.  Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections , 2007, Journal of Clinical Virology.

[44]  Shigeyuki Yokoyama,et al.  An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo[(4,5)-b]pyridine. , 2003, Journal of the American Chemical Society.

[45]  E. Ohtsuka,et al.  A new, but old, nucleoside analog: the first synthesis of 1-deaza-2'-deoxyguanosine and its properties as a nucleoside and as oligodeoxynucleotides. , 2003, Nucleic acids research.

[46]  F. Romesberg,et al.  Unnatural substrate repertoire of A, B, and X family DNA polymerases. , 2008, Journal of the American Chemical Society.

[47]  S. Yokoyama,et al.  A two-unnatural-base-pair system toward the expansion of the genetic code. , 2004, Journal of the American Chemical Society.