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The synthesis and insertions of ( S )-1- O -(pyren-1-ylmethyl)glycerol into intercalating nucleic acids is described. Insertions of this S -isomer as a bulge lead to reduced binding affinity towards complementary ssDNA compared to intercalating nucleic acids possessing ( R )-1- O -(pyren-1-ylmethyl)glycerol in the same positions. Insertions of both ( R ) or ( S ) 1- O -(pyren-1-ylmethyl)glycerols as bulges into two complementary strands decreased the stability of the complex compared to dsDNA possessing the pyrene pseudo-nucleotide in one of the strands.

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C(1)T(2)C(3)A(4)A(5)C(6)X(7)C(8)A(9)A(10)G(11)C(12)T(13)):d(A(14)G(15)C(16)T(17)-T(18)G(19)X(20)G(21)T(22)T(23)G(24)A(25)G(26)). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used two-dimensional (1)H NMR spectroscopy to determine a high-resolution solution structure of the weak INA-INA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 A. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.

Fluorescence and hybridization specificity is reported for intercalating nucleic acids (INAs), which are here oligodeoxynucleotides (ODNs) synthesized with insertions using (S)-1-[bis(4-methoxyphenyl)(phenyl)methoxy]-3-[(pyren-1-yl)methoxy]propan-2-ol phosphoramidites. It is shown that an INA with two insertions placed as next-nearest neighbors can be used for discrimination between nucleic acids and their single-point mutants. Quenching of an excimer band at 480 nm is observed upon hybridizing to a complementary sequence, whereas the excimer band is present when the nucleobase pair between the two pyrene moieties is mismatched. It is the first example of a solution based on fluorescence detection of single-point mutants that uses excimer formation and does not rely on stringent hybridization conditions. Furthermore, it is shown that INAs with pyrene insertions retain their sequence specificity in thermal melting.

This chapter describes two different fast protocols for optimizing EasyBeacons for the challenging detection of the methylation status of a single CpG duplet in bisulfitetreated DNA. EasyBeacons can be used in multiplex detections even if they do not have the same affinity for their respective targets giving very specific results for both realtime polymerase chain reaction (real-time PCR) detection and endpoint analysis using inexpensive master mixes. The technology described in this chapter is a very competitive alternative to other real-time PCR technologies.

We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4'-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9 degrees C per modification when the IPN was added as a dangling end and up to 6.7 degrees C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8 degrees C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.