modified nucleic acids have great potential for applications in oligonucleotide - based drug design . as natural rna and
dna molecules are highly sensitive towards nuclease digestion and often possess low thermal stability , great effort has been made to design nucleic acid modifications that stabilize rna or dna while simultaneously maintaining the overall watson - crick base pairing ability .
modified nucleic acids are indispensable for future applications comprising diagnostic and clinical approaches like the use of aptamers or the sirna technology . extensive and challenging experiments and investigations
have been undertaken to develop nucleotide analogues that maintain the overall a - rna - type conformation and n - type sugar puckering , as such modifications are likely to allow the substitution of rna without large changes in functionality .
considerable effort has been made in the synthesis and characterization of 2-o - methyl - rnas , 2-f - rnas , phosphoramidate - rnas , and the  locked  nucleic acid family . by using locked nucleotide building blocks containing the 2-o,4-c - methylene--d - ribofuranose ( lna ) modification , a significant increase in thermostability can be observed in accordingly substituted rnas . for example , the melting temperature of modified rna helices can be increased by + 2 to + 10c per lna monomer substitution . to understand the stabilizing effects of lna - substituted rnas ,
numerous structural investigations have been performed during the past years to investigate their conformation in detail .
these studies provided insights in the local geometric parameters of mix - mer lna - rna helices and of lna - rna heteroduplexes .
the 2-o,4-c - methylene--d - ribofuranose lna - rna mix - mer duplexes maintain mainly the overall a - type nucleic acid conformation . on the other hand ,
the 2-o,4-c--l - ribofuranose lna modification is used in dna substitution , as this modification preserves the overall b - type nucleic acid geometry of dna .
thus , there are two powerful nucleotide modifications with great potential in drug design , the 2-o,4-c - methylene--d - ribofuranose nucleotides ( lna ) for rna substitution and the 2-o,4-c - methylene--l - ribofuranose nucleotides to modify dna .
the structure of heteroduplexes , consisting of one fully modified lna strand hybridized to either rna or to dna , revealed the following : the rna conformation is maintained upon hybridizing a 2-o,4-c - methylene--d - ribofuranose lna strand to rna , whereas a mixed n- and s - type sugar puckering is induced by hybridizing a 2-o,4-c - methylene--d - ribofuranose lna to dna . the b - type conformation is maintained by using a 2-o,4-c - methylene--l - ribofuranose lna strand targeted to dna .
it is generally accepted that the 2-o,4-c - methylene--d - ribofuranose  locks  the lna in the c3-endo conformation .
this approach is used to direct the geometry of the phosphate backbone in a manner to orient the duplex towards a more efficient base stacking . even though the 2-o,4-c - methylene--d - ribofuranose lna - rna mix - mer duplexes maintain the overall a - type nucleic acid conformation , molecular dynamics simulations   and a crystal structure   of  all  lna duplexes , consisting exclusively of 2-o,4-c - methylene--d - ribofuranose building blocks , yielded insights into a novel nucleic acid geometry .
an  all lna  duplex shows alterations in the local and overall helical parameters as compared to natural rna and can rather be compared to other modified nucleic acids , like glycol nucleic acids ( gnas ) , peptide nucleic acids ( pnas ) , or homo - dna
. an lna duplex appears as a right - handed , antiparallel helix that maintains the canonical watson - crick base pairing and the 2-exo conformation for all nucleotides .
nevertheless , the lna duplex shows a considerable decrease in the helical twist , roll and propeller twist , which facilitates a widening of the major groove and a decrease of the minor groove dimensions .
these alterations induce a large hollow cave in the middle of the duplex that is obvious in a projection perpendicular to the helical axis . due to an enlarged helical rise and the unwinding of the helix , which results from the decrease in the twist angle parameters , the lna duplex possesses an increased helical pitch .
the unique nucleic acid geometry of  all lna  helices apparently induces a more efficient and stable base stacking , which contributes to the higher thermostability of lnas and lna - modified nucleic acids .
interestingly , the structure of an rna / lna heteroduplex   is a geometric intermediate between the rna and the  all lna  conformation .
the 7mer lna helix was derived from the e. coli trna isoacceptor with the data base  compilation of trna sequences and sequences of trna genes 
i d rs 1661   and represents the sequence of the trna aminoacyl stem microhelix that has been crystallized previously possessing the sequence 5-(g - g - u - g - a - g - g-)-3 and 5-(c - c - u - c - a - c - c-)-3 .
the base sequence of the rna was maintained for further comparative studies , except for the u to t and the c to mc exchange used in standard lna synthesis .
the chemically synthesized single strands with the sequences 5-(g - g - t - g - a - g - g)-3 and 5-(mc - mc - t - mc - a - mc - mc)-3 were purchased from iba ( gttingen , germany ) with hplc purification grade .
crystals were grown within 3 - 4 days using 40  mm sodium cacodylate , ph 5.5 , 20  mm cobalt hexamine , 80  mm sodium chloride , 20  mm magnesium chloride , and 10% ( v / v ) mpd with equilibration against 1  ml 3341% ( v / v ) mpd at 21c using the hanging drop vapour diffusion technique .
data collection of the lna crystals was performed at the elettra synchrotron ( trieste , italy ) beam line xrd-1 at a wavelength of 1.0   and a temperature of 100  k. the crystal diffracted up to 1.9   resolution .
the corresponding trna - microhelix was measured at the desy synchrotron ( hamburg , germany ) at a wavelength of 0.8123   , 100  k temperature , and diffracted up to 1.2   .
molecular replacement calculations were performed using the program phaser   within the ccp4i program suite .
the lna structure was solved by using a model built from the previously solved trna microhelix structure but exchanging the riboses by 2-o,4-c - methylene--d - ribofuranose residues .
standard lna nucleotides were used for model building , which comprises the standard u to t and c to mc substitutions in lna as compared to rna .
refinement calculations were done applying the program refmac , and electron density maps were calculated using fft , as implemented in the ccp4i package .
structure representations and graphical analysis of helices were performed with the programs coot   and pymol .
we analysed the crystal structure of a  locked  nucleic acid duplex , which contains exclusively 2-o,4-c - methylene--d - ribofuranose nucleotides ( figure 1 ) , in comparison to the structures of the naturally occurring rna as well as to other backbone - modified nucleic acids like glycol nucleic acids ( gnas ) or peptide nucleic acids ( pnas ) .
the lna helix structure reveals a nucleic acid duplex geometry that significantly differs from the canonical a - type rna structure ( figure 2 , tables 2 and 3 ) .
the structure of the lna duplex appears as a stretched helical ladder with altered local and overall geometric parameters .
the observed geometry can be rather compared to that of glycol nucleic acids ( gnas ) , peptide nucleic acids ( pnas ) [ 26 , 27 ] , or homo - dnas .
we detected a notable decrease in several local and overall helical parameters in the lna helix , like the twist , roll and propeller twist , as compared to a corresponding rna molecule ( table 2 ) .
this results in a widening of the major groove , a decrease in helical winding and an increased helical pitch .
the major groove dimensions in the lna duplex showed values of around 24 - 25   in diameter , as compared to 16   observed for the canonical a - rna duplex .
concomitantly , the minor groove of lna duplexes is narrower ( about 15   ) than that of standard rna helices ( 19   ) . on the other hand , the slide and rise values
are slightly increased in the lna helices . moreover , the shift of the base pairs in the lna duplex results in an empty tunnel running through the center of the helix . in the lna helix , the low twist angle of 26 and the large pitch of 14 base pairs per turn lead to an unwinding of the duplex , as compared to rna , which possesses a twist of 32 and a pitch of 11 base pairs per turn .
the helical rise of lna falls into a range of 2.83.0   , whereas the helical rise in rna is 2.6   . due to the increased rise and the unwinding of the helix , the lna possessed an enlarged helical pitch of 39   , as compared to 29   in rna helices .
the backbone torsion angles resembled the sc , ap , sc , sc ap , sc , and ap conformation for the  ,  ,  ,  ,  ,  , and  angles with the sugar puckering being in the 2-exo conformation .
the phosphate - phosphate distances are in the region of 5.6   as compared to 6.0   for rna .
it is conceivable that the altered helical parameters in lna duplexes provide an enhancement in nucleotide stacking , leading towards stronger - interactions of the base pairs .
it is well accepted that the extensive hydration of the rna minor groove plays an important role in the structure / function relationship .
as the specific hydration pattern of rna is governed by the 2-hydroxyl group , it has been questioned whether the 2-o,4-c - methylene--d - ribofuranose in lna allows a comparable hydration as described for rnas .
therefore , we focussed our investigations on analyzing the arrangement of the solvent molecules surrounding the lna duplex .
we observed that the distribution of water molecules in the lna minor groove follows the general pattern known for rna hydration , as the bridged 2 oxygen atoms in the 2-o,4-c - methylene--d - ribofuranose moieties serve as hydrogen bond acceptors similar to the 2-hydroxyl residues in rna .
interestingly , the structure of an rna / lna hybrid helix represents a geometric intermediate between rna and  all lna  helices . in figure 4
, we present the structure of idealized rna as compared to the rna / lna hybrid ( pdb i d : 1h0q ) and to the  all lna  duplex ( pdb i d : 2x2q ) as calladine - drew plot . to better visualize the overall geometry in this figure ,
the helices were extended to 22 base pairs for rna , to 25 base pairs for rna / lna , and to 28 base pairs for lna showing two full helical turns each . for an overall comparison of a - rna and b - dna helices to the conformation of different backbone - modified nucleic acid types , like gna and pna ( figure 5 )
, we displayed the selected nucleic acid helices with a total length of 46 base pairs ( figure 6 ) .
we illustrate the natural dna and rna and the synthetic gna ( pdb i d : 2jja ) and pna ( pdb i d : 1pup ) as compared to the lna ( pdb i d : 2x2q ) duplex structure . the standard a- and b - type nucleic acid conformations are paraphrased by the rna and dna helices .
the gna shows the structure of a helical ribbon with only one large minor groove and completely lacks the major groove , which is instead a convex surface .
the pna resembles the helix with a wide and deep major groove concomitant with a narrow and shallow minor groove [ 26 , 27 ] .
the weakly twisted right - handed homo - dna structure has been described to explain the inability of allo- , altro- , and glucanosyl - nucleotides to form stable base pairing systems ( picture not shown ) . in the middle of figure 6 we present the extended structure of the lna duplex , which represents the unusual geometry , which can rather be brought into vicinity of gna , pna , and homo - dna than to the natural nucleic acid duplexes dna and rna .
the lna helix , however , possesses a pitch of 39   with 14 base pairs per helical turn and an average rise of 2.8   .
dna and rna show an average pitch of 30   and 34   , respectively , as compared to mean values of 60   , 58   , and 53   found for gna , pna , and homo - dna .
( figure 6 , table 3 , no data shown for homo - dna ) . conclusively , regarding the geometry of the lna duplex in total , this helix resembles a more natural nucleic acid , when compared to the other backbone - modified duplexes . as has been reviewed , an increase of the melting temperature between + 2 to + 10c can be observed per lna building block added in strands hybridized to rna .
the short 7  bp lna duplex , derived from the trna microhelix for this study , exhibits a melting temperature of above 90c , whereas the corresponding rna has a tm value of 45.0c .
we have previously investigated the melting temperature of another lna 7mer helix in comparison to its natural rna counterpart .
similarly , the lna duplex possesses a tm value of 84.3c , whereas its corresponding rna helix melts at 22.4c . the drastic shift in thermostability in both lnas
as compared to the rnas corresponds to an average of 4.5c per nucleotide building block , which is consistent with the reviewed observations .
thus , the thermostability data and the structure properties of lnas provide new perspectives for future nucleic acid drug applications , which is an encouraging outlook . considering that the increase in tm values by substituting natural nucleic acids with single or multiple nucleotides by lna residues seems to be a summative property
nearly any natural nucleic acid can be modified by introducing single and multiple lna building blocks or even complete lna duplexes , thereby stepwise increasing the thermostability depending on the number of introduced lna residues .
depending on the particular requirements , any nucleic acid can thus be stabilized at will with little or no loss of function . in this respect ,
lna substitution may serve as a reliable method to stabilize nucleic acids , in particular aptamers , for clinical applications .
an upcoming challenge is to stabilize aptamer stem regions by introducing lna portions without affecting the loop regions that are usually essential for target binding and specificity .
several reports in the literature highlight a forthcoming application of lna - substituted aptamers with retained or even improved ligand - binding capacity .
an exciting example is the use of lna modifications within hammerhead ribozymes that improve the overall cleaving capacity .
in addition , lna modifications have successfully been introduced into antisense oligonucleotides and dnazymes that were targeted to functionally selected binding sites and inhibited hiv-1 expression .
a third example describes a g - quadruplex thrombin aptamer , which retained the biological activity to a varying extent depending on the nucleotide positions that were lna modified .
these selected reports are a snapshot of numerous studies demonstrating the great potential of lna substitutions in functional nucleic acids and possible therapeutic applications . the crystal structure of the  all locked 
nucleic acid helix contributes to the understanding of the structure / function relationship and the high thermostability of these molecules . in summary lnas
possess an encouraging potential for the development of new stabilized nucleic acids and will promote future applications in diagnostics , drug discovery , and clinical therapy .
  locked nucleic acids  ( lnas ) belong to the backbone - modified nucleic acid family . 
 the 2-o,4-c - methylene--d - ribofuranose nucleotides are used for single or multiple substitutions in rna molecules and thereby introduce enhanced bio- and thermostability . 
 this renders lnas powerful tools for diagnostic and therapeutic applications . 
 rna molecules maintain the overall canonical a - type conformation upon substitution of single or multiple residues / nucleotides by lna monomers . 
 the structures of  all  lna homoduplexes , however , exhibit significant differences in their overall geometry , in particular a decreased twist , roll and propeller twist . 
 this results in a widening of the major groove , a decrease in helical winding , and an enlarged helical pitch . 
 therefore , the lna duplex structure can no longer be described as a canonical a - type rna geometry but can rather be brought into proximity to other backbone - modified nucleic acids , like glycol nucleic acids or peptide nucleic acids . 
 lna - modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery . 
1. Introduction
2. Material and Methods
3. Results and Discussion