Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers b-L-LNA and a-D-LNA; studies in the mirror image world Nanna K. Christensen, a Torsten Bryld, a Mads D. Sørensen, b Khalil Arar, c Jesper Wengel a and Poul Nielsen* a a Nucleic Acid Center†, Department of Chemistry, University of Southern Denmark, DK-5230 Odense, Denmark. E-mail: pon@chem.sdu.dk b Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark c Proligo, Rue Delaunay 1, Paris 75011, France Received (in Cambridge, UK) 3rd October 2003, Accepted 3rd December 2003 First published as an Advance Article on the web 19th December 2003 Two LNA (locked nucleic acid) stereoisomers (b-L-LNA and a- D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and a-L-LNA and their L- DNA and L-RNA complements. Both are found to display high- affinity RNA-recognition by the formation of duplexes with parallel strand orientation. Conformationally restricted oligonucleotides have enabled high affinity recognition of DNA and RNA. 1,2 In the LNA-family of stereoisomeric locked nucleic acid analogues the nucleoside monomers are locked in N-type conformations (Fig. 1), 3–10 and both LNA 3–5 and a-L-LNA sequences 6–8 (i.e. LNA with b-D- and a-L-configurations, respectively)‡ have demonstrated unprece- dented antiparallel hybridisation with both DNA and RNA complements. This duplex stabilisation is also evident for mixmers of LNA or a-L-LNA nucleotides and natural 2A-deoxyribonucleo- tides. In order to investigate the scope of parallel nucleic acid recognition we recently introduced a-LNA (or a-D-LNA; LNA with a-D-configuration).‡ 9,10 The formation of parallel duplexes has been reported for a-DNA (i.e. the a-anomer of DNA) with complementary DNA and RNA, 11–13 and subsequently, mixed fully modified pyrimidine a-LNA sequences were found to recognise complementary RNA, but not DNA, forming strong parallel stranded duplexes. 10 With the furanose rings locked in N-type (C-3A-endo) conforma- tions, LNA is essentially a perfect RNA-mimic, 3–5 whereas the situation for a-LNA is more complicated. Thus, in LNA–DNA mixmers, the LNA-monomers have been found to tune the neighbouring DNA-monomers towards N-type conformations thereby inducing the formation of overall A-type duplexes. 14 On the other hand, it is unlikely that a-configured nucleosides exist in a perfect N-type conformation due to the reverse influence of the anomeric effect, and a-LNA monomers are unable to tune neighbouring a-DNA monomes towards N-type conformations. Furthermore, a-LNA is not an obvious conformational mimic of either a-DNA or a-RNA. NMR studies of duplexes containing a-L-LNA sequences and complementary DNA or RNA have led to the conclusion that this LNA stereoisomer can be regarded as a DNA-mimic. 15,16 With LNA being an RNA mimic and a-L-LNA being a DNA mimic, we deduce the “a-anomer” of a-L-LNA, i.e. b-L-LNA, to be an a- DNA mimic and subsequently an even stronger candidate for parallel nucleic acid recognition than a-LNA. In this communica- tion we explore this hypothesis by comparing the hybridisation properties of b-L-LNA and a-LNA sequences of mixed base composition. However, the synthesis of b-L-LNA monomers has not been realised, and the studies were performed with LNA and a- L-LNA in the mirror-image world. We have previously studied oligothymidylate sequences by this strategy 17 but here we introduce mixed sequences allowing conclusions about general hybridisation behaviour including strand orientation. A decamer a-L-LNA sequence (Table 1) was prepared from the appropriate thymine, adenine and 5-methylcytosine phosphor- amidite building blocks 8 on a universal support in order to obtain a completely modified a-L-LNA sequence. The LNA-sequence of the same base composition was obtained in a similar way by a standard LNA-synthesis protocol. 4 Four complementary L-DNA and L-RNA sequences were designed as both parallel and antiparallel complements as well as with single A/T or A/U mismatches.§ Standard DNA and RNA sequences were used as reference strands (Table 1). The applied standard sequence was designed as a non-self-complementary sequence. As expected, both the a-L-LNA sequence and the LNA sequence were found to recognise their antiparallel DNA and RNA complements with very high affinity (T m = 66–87 °C, Table 1) and with the expected selectivity for match over mismatch sequences (DT m = 216 °C). With parallel complements, the situation was more complicated. Thus, complexes with mismatch sequences were more stable than with match sequences with T m As up to 51 °C for the LNA:RNA complex. However, we deduce these complexes to be antiparallel wobble structures rather than regular parallel duplexes. When the a-L-LNA sequence was mixed with com- plementary L-configured DNA and RNA sequences, the observa- tions earlier made for an a-LNA pyrimidine sequence 10 were supported. Thus, no complex could be detected with either the antiparallel complements or with parallel complementary DNA. With the parallel RNA complement, a melting temperature of 44 °C was observed. When the LNA sequence was mixed with the L- configured complements, the general properties of the new b-L- LNA analogue were examined. No complexes were detected with antiparallel DNA and RNA complements, whereas stable duplexes with both parallel DNA and RNA (with almost identical thermal stabilities, 42 °C and 44 °C, respectively) were formed. The base- pairing selectivity, which was questioned in our first study on an oligothymidylate sequence, 17 was here confirmed to be satisfactory (215 °C and 212 °C, respectively) for a mixed sequence. Thus, b-L-LNA and a-LNA demonstrate equal strength in parallel RNA-recognition, but only the former forms a duplex with † Nucleic Acid Center is funded by the Danish National Research Foundation for studies on nucleic acid chemical biology. Fig. 1 Structures of LNA-stereoisomers. This journal is © The Royal Society of Chemistry 2004 DOI: 10.1039/b312321a 282 Chem. Commun., 2004, 282–283