DOI: 10.1002/chem.201002109 C5-Functionalized DNA, LNA, and a-l-LNA: Positional Control of Polarity- Sensitive Fluorophores Leads to Improved SNP-Typing** Michael E. Østergaard, [a] Pawan Kumar, [a, b] Bharat Baral, [a] Dale C. Guenther, [a] Brooke A. Anderson, [a] F. Marty Ytreberg, [c] Lee Deobald, [d] Andrzej J. Paszczynski, [d] Pawan K. Sharma, [b] and Patrick J. Hrdlicka* [a] Introduction Single nucleotide polymorphisms (SNPs) are the most fre- quently occurring genetic variation in the human genome (> 9 million SNPs, one SNP per 1000 base pairs). [1] SNPs often result in phenotypic changes, and are accordingly im- portant markers in disease genetics and pharmacogenomic studies. The most established SNP genotyping technologies are enzyme-based or rely on small differences in thermosta- bility between duplexes of probes and complementary or SNP-containing targets. [1, 2] Moreover, these multistep proto- cols often necessitate stringent control of assay conditions (e.g., temperature, ionic strength). As a result, there has been a major thrust to develop alternative SNP-typing ap- proaches, which are operationally more simple and cost-effi- cient. Examples include modified molecular beacons, [3] dual probes, [4] quenched autoligation probes, [5] intercalator-modi- fied probes, [6] charge transfer based approaches, [7] and base- discriminating fluorescent (BDF) probes. [8] Oligodeoxyribo- nucleotides (ONs) modified with 5-[3-(1-pyrenecarboxami- do)propynyl]-2’-deoxyuridine monomer X (Figure 1) have emerged as particularly promising BDF probes due to their efficient optical discrimination of complementary over mis- matched targets and moderately high fluorescence quantum yields, [8d] which has enabled discrimination of SNPs in human breast cancer cell lines at 50 nm target concentra- tion. [9] Molecular modeling and photophysical studies sug- gest that the polarity-sensitive 3-(1-pyrenecarboxamido)pro- pynyl moiety of monomer X (Figure 1) intercalates into the hydrophobic base stack upon hybridization with mismatched targets resulting in fluorescence quenching, while it points toward the polar major groove in duplexes with complemen- tary DNA targets resulting in high fluorescence. [8d] These differences in binding modes are suggested to correlate with changes in the glycosidic torsion angle (O4’-C1’-N1-C2) from anti to syn ranges. Abstract: Single nucleotide polymorph- isms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5-[3-(1-pyrenecar- boxamido)propynyl]-2’-deoxyuridine monomer X enable detection of SNPs at non-stringent conditions due to dif- ferential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal de- naturation and optical spectroscopic characteristics of monomer X are com- pared to the corresponding locked nu- cleic acid (LNA) and a-l-LNA mono- mers Y and Z. ONs modified with monomers Y or Z result in a) larger in- creases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluores- cent duplexes due to markedly larger fluorescence emission quantum yields (F F = 0.44–0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of mono- mers X–Z adopt anti and syn confor- mations upon hybridization with matched and mismatched targets, re- spectively. The polarity-sensitive 1-pyr- enecarboxamido fluorophore is, there- by, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and a-l-LNA mono- mers Y and Z influence the glycosidic torsional angle profile leading to al- tered positional control and photophys- ical properties of the C5-fluorophore. Keywords: bicyclic nucleotides · BNA · DNA targeting · pyrene · single nucleotide polymorphisms [a] Dr. M. E. Østergaard, Dr. P. Kumar, B. Baral, D. C. Guenther, B. A. Anderson, Prof. P. J. Hrdlicka Department of Chemistry, University of Idaho P.O. Box 442343, Moscow, ID 83844-2343 (USA) Fax: (+ 1) 208-885-6173 E-mail: hrdlicka@uidaho.edu [b] Dr. P. Kumar, Prof. P.K. Sharma Department of Chemistry, Kurukshetra University Kurukshetra 136119 (India) [c] Prof. F. M. Ytreberg Department of Physics, University of Idaho P. O. Box 440903, Moscow, ID 83844-0903 (USA) [d] Dr. L. Deobald, Prof. A. J. Paszczynski Environmental Biotechnology Institute, University of Idaho P. O. Box 441052, Moscow, ID 83844-1052 (USA) [**] LNA = locked nucleic acid; SNP = single nucleotide polymorphism. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201002109. Chem. Eur. J. 2011, 17, 3157 – 3165  2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 3157 FULL PAPER