nucleic acid systems have proven to be very amenable to the design of nanostructures . while there are more published examples for dna systems ( 14 ) , rna has also been used to self - assemble into various shapes like squares and triangles ( 4,5 ) . the corners of the assembled rna complexes are based on known helical junctions ( 4 ) or loop  loop interactions ( 6 ) . these designed rna structures highlight the importance of loop  loop motifs and helical junctions . it has been shown , for example , that a four - way junction promotes the functional folded state of the hairpin ribozyme ( 7 ) . thus , characterizing and classifying rna junctions can lead to a better understanding of the structural and functional capabilities of rna . the basic repository for experimentally determined nucleic acid structures is the protein data bank ( pdb ) ( 8) . because rna structures are only part of the content of the pdb , several databases provide additional information by annotating and classifying rna structures derived from the pdb . scor is a structural database that contains a classification of internal and hairpin loops ( 911 ) . its classification scheme is based on a directed acyclic graph , allowing a node to have multiple parents . ncir is a database of non - canonical interactions found in rna structures ( 12 ) . for each base pair type , ncir provides information about sequence and structure contexts in which this base pair type has been found . the nucleic acid database ( ndb ) is a database containing annotated and categorized rna and dna structures ( 13 ) . among other things the metals in rna ( merna ) database catalogs rna structures that are bound to metal ions ( 14 ) . lescoute and westhof ( 15 ) analyzed rna structures with respect to three - way junctions . they found that three - way junctions that contain two helices that are coaxially stacked can be classified into three main families depending on the relative lengths of the connecting loop regions . lilley et al . ( 17 ) describe a nc - iubmb recommended nomenclature for nucleic acids junctions . we have developed a database called rnajunction that provides information about rna junctions , kissing loops , internal loops and bulges in an extracted , annotated and searchable form . our rnajunction database is very useful for analyzing and understanding the principles of rna structure formation . it is to our knowledge the only currently available database that also contains extracted rna kissing loop elements . its unique search capability allows the user to identify rna junctions based on ( among other criteria ) inter - helical angles , which makes it an important resource for the design of novel rna nanostructures from building blocks . our definition of an n - way rna ( or dna ) junction is best explained with the help of figure 1 , a schematic view of a hypothetical three - way junction . one can see from that example that an extracted n - way junction consists of n strands and n connector helices . note that the original structure from which the junction is extracted might consist of only one strand . also , internal loops and bulges can be viewed as special cases of  two - way junctions . our definition of rna helix junctions is geared towards the design of rna nanostructures . from this standpoint , junctionscanner, for detecting , extracting and analyzing rna junctions , kissing loops , internal loops and bulges from pdb coordinate files . the algorithm uses the rnaview ( 18 ) base pairing patterns to detect groups of interconnected helices corresponding to rna junctions from a given structure in pdb format . given the list of base pairs , a list of all bulge - free helices is generated ( a helix containing a bulge is represented as two helices ) . path as follows : using a start strand and helix position , the strand is followed in the 5 to 3 direction to the next downstream helix . the opposing strand from this subsequent helix is now followed from its 5 to its 3 end to yet another helix and so forth . if one arrives in this fashion back at the helix one started with ( a circular path ) , one can conclude that the helices of the path form a junction ( compare figure 1 ) . also , the local orientation of each helix is approximated by superposing a helix represented by the idealized positions of c4 atoms ( 19 ) . this permits the computation of the angle between the direction - vectors of any two helices . we found that the number of extracted structural elements depends on the cutoff - parameters used by the various filters . depending on the use , one wants to apply strict or relaxed filter parameters . to account for different applications , we generated three different sets of extracted structural elements using parameter sets ranging from least strict to  most strict ( the values of the different parameter sets are shown in table 1 ) . the connector helices have to consist of at least n base pairs ( n being two , three or four , table 1 ) . an idealized helix has to be fitted onto the connector helices with an rmsd being smaller than the chosen cut - off of 2.5 or 3.0  ( table 1 ) . the sum of the number of nucleotides of the junction loop regions may not exceed 50 nt . another optional filter is the  corridor - filter : corridors are defined as cylindrical regions ( 3  radius ) with their base being located at the center of the junction 's helix ends . the cylinders are oriented such that they point outward in the direction of the respective helix ( the geometry involved in the corridor filter is shown schematically in figure s2 in the supplementary data ) . this steric clash check limits the retrieved junctions to cases that are potentially useful for the  mosaic unit approach of building larger structures from building blocks ( 2022 ) . medium and  least strictmost strictmediumleast strictminimum helix length ( bp)432rms for helix fit ( )2.53.03.0corridor filteryesyesnonote that the structural elements corresponding to the most strict parameter set are required to possess helices of lengths four ( or greater ) in the original structure , however the corresponding extracted coordinate files contain only three base pairs . medium and  least strict note that the structural elements corresponding to the most strict parameter set are required to possess helices of lengths four ( or greater ) in the original structure , however the corresponding extracted coordinate files contain only three base pairs . the detection of kissing loops is handled in a manner that is similar to junction detection . again , in a first step a set of all rna double helices is generated . each set of three helices that interact according to the connectivity shown in figure 2 is considered a kissing loop interaction . using the schematic drawing of figure 2 as an example , the loop region of helix h1 consists in our definition of the loops l11 , l12 as well as one strand of helix h3 . for each pair of stems a and b , the algorithm tries to find a third helix c , that is located in the loop region of stems a and b. to reduce the computational cost , we applied further constraints by requiring that the helix ends of helices a and b must be closer than 50  . each circle represents one nucleotide ; the single - stranded regions are indicated in gray . each circle represents one nucleotide ; the single - stranded regions are indicated in gray . we downloaded 1176 structures from the pdb database that contain rna ( as of june 2007 ) . the base pairing patterns were ascertained using rnaview ( 18 ) . the junctionscanner program described in the previous section was used to parse the pdb and rnaview data . for each identified structural element the junctionscanner program generates two files : a file containing the extracted element in pdb format , and a text file describing all identified properties ( such as helix orientation , inter - helix angles , nomenclature , nucleotide sequences , residue indices ; the full list of properties is shown in the supplementary data ) . the junctionscanner algorithm was applied five times to each coordinate input file to avoid the unlikely chance that a helix was missed due to the random component inherent in the helix - fitting algorithm . the algorithm identified 258 kissing loop structures , 9357 internal loops and bulges , 2065 three - way junctions , 1091 four - way junctions , 462 five - way junctions , 70 six - way junctions , 23 seven - way junctions , one eight - way junction and one nine - way junction . five hundred and fifty - nine different pdb files contained structural elements meeting the minimum filter criteria described earlier . the counts for the number of identified junctions depend on how strict the parameters for the junction - detection algorithm are chosen . counts for the different junction types and junction scanner parameter sets are listed in table 2 . if a structural element is identified using several different filter parameter sets , only the version corresponding to the strictest parameter set is stored in the database , thus avoiding unnecessary redundancy . table 2.counts of identified rna junctions , internal loops , bulges and kissing loops the counts are given for three different filter parametersstructural elementmost strictmedium strictleast strictreduced redundancykissing loop9820625851internal loop / bulge31335635935718373-way junction731141220654314-way junction28049010912725-way junction109237462976-way junction4670177-way junction455561168-way junction01119-way junction0011the last column shows the counts for the reduced - redundancy least - restricted data set whose representative structures can be part of either the most - strict , medium - strict or least - strict data set . counts of identified rna junctions , internal loops , bulges and kissing loops the counts are given for three different filter parameters the last column shows the counts for the reduced - redundancy least - restricted data set whose representative structures can be part of either the most - strict , medium - strict or least - strict data set . all structural elements of the same type , consisting of the same sequences were clustered using single linkage clustering and a structural superposition cutoff of 3.0  drms ( c4 atom positions ) . for each cluster , one of its members this reduces the number of structural elements by a factor of about four ( compare table 2 ) . analyzing the structural variation within clusters , we found that in all but three cases the drms of the cluster members with respect to their cluster representative is smaller then 3.0  . the results of the junctionscanner applied to pdb coordinate files was entered into a mysql relational database . these data include the quantitative analysis , sequence , geometric conformation and citation for the identified structural elements as well as references to the parent structure from the pdb and the extracted coordinate data ( the full list of stored properties is given in the supplementary data ) . images of the secondary structure ( generated using rnaview ) and tertiary structure [ generated using raster3d ( 23 ) ] are available . the 3d coordinates of the structural element can be viewed interactively using the jmol java applet . the text output of the junctionscanner run is also provided and contains more detailed information such as the local coordinate systems of the fitted idealized helices . additional data is included in the form of structures obtained by molecular mechanics minimization , calculated using amber 8.0 ( 24 ) and the cornell force field ff99 ( 25 ) . analyzing the minimized structures , we found that 68% of them have an rmsd of < 1  compared to their respective original structure ; 31% have an rmsd between 1 and 2  , 0.7% exhibit an rmsd between 2 and 3  , 0.0003% show an rmsd between 3 and 5  . we plan to supplement the rnajunction database also with molecular dynamics simulation results in the future . the essential tables underlying the rnajunction database correspond to junctions , helices , strands , references and angles . in this way , helices and junctions , for example , are related through a many - to - one relationship , allowing in principle for junctions of arbitrarily large order . more detailed information about the design of the database tables is provided in the supplementary data . internal loops , bulges and kissing loops are internally considered a special case of  two - way junctions ; a flag indicates the different strand connectivity of a kissing loop compared to an internal loop . our definition of an n - way rna ( or dna ) junction is best explained with the help of figure 1 , a schematic view of a hypothetical three - way junction . one can see from that example that an extracted n - way junction consists of n strands and n connector helices . note that the original structure from which the junction is extracted might consist of only one strand . also , internal loops and bulges can be viewed as special cases of  two - way junctions . our definition of rna helix junctions is geared towards the design of rna nanostructures . from this standpoint , junctionscanner, for detecting , extracting and analyzing rna junctions , kissing loops , internal loops and bulges from pdb coordinate files . the algorithm uses the rnaview ( 18 ) base pairing patterns to detect groups of interconnected helices corresponding to rna junctions from a given structure in pdb format . given the list of base pairs , a list of all bulge - free helices is generated ( a helix containing a bulge is represented as two helices ) . path as follows : using a start strand and helix position , the strand is followed in the 5 to 3 direction to the next downstream helix . the opposing strand from this subsequent helix is now followed from its 5 to its 3 end to yet another helix and so forth . if one arrives in this fashion back at the helix one started with ( a circular path ) , one can conclude that the helices of the path form a junction ( compare figure 1 ) . also , the local orientation of each helix is approximated by superposing a helix represented by the idealized positions of c4 atoms ( 19 ) . this permits the computation of the angle between the direction - vectors of any two helices . we found that the number of extracted structural elements depends on the cutoff - parameters used by the various filters . depending on the use , one wants to apply strict or relaxed filter parameters . to account for different applications , we generated three different sets of extracted structural elements using parameter sets ranging from least strict to  most strict ( the values of the different parameter sets are shown in table 1 ) . the connector helices have to consist of at least n base pairs ( n being two , three or four , table 1 ) . an idealized helix has to be fitted onto the connector helices with an rmsd being smaller than the chosen cut - off of 2.5 or 3.0  ( table 1 ) . the sum of the number of nucleotides of the junction loop regions may not exceed 50 nt . another optional filter is the  corridor - filter : corridors are defined as cylindrical regions ( 3  radius ) with their base being located at the center of the junction 's helix ends . the cylinders are oriented such that they point outward in the direction of the respective helix ( the geometry involved in the corridor filter is shown schematically in figure s2 in the supplementary data ) . this steric clash check limits the retrieved junctions to cases that are potentially useful for the  mosaic unit approach of building larger structures from building blocks ( 2022 ) . medium and  least strictmost strictmediumleast strictminimum helix length ( bp)432rms for helix fit ( )2.53.03.0corridor filteryesyesnonote that the structural elements corresponding to the most strict parameter set are required to possess helices of lengths four ( or greater ) in the original structure , however the corresponding extracted coordinate files contain only three base pairs . medium and  least strict note that the structural elements corresponding to the most strict parameter set are required to possess helices of lengths four ( or greater ) in the original structure , however the corresponding extracted coordinate files contain only three base pairs . the detection of kissing loops is handled in a manner that is similar to junction detection . again , in a first step a set of all rna double helices is generated . each set of three helices that interact according to the connectivity shown in figure 2 is considered a kissing loop interaction . using the schematic drawing of figure 2 as an example , , l12 as well as one strand of helix h3 . for each pair of stems a and b , the algorithm tries to find a third helix c , that is located in the loop region of stems a and b. to reduce the computational cost , we applied further constraints by requiring that the helix ends of helices a and b must be closer than 50  . the other filters used for junctions are also applied . 
 figure 2.schematic drawing of a kissing loop . each circle represents one nucleotide ; the single - stranded regions are indicated in gray . each circle represents one nucleotide ; the single - stranded regions are indicated in gray . we downloaded 1176 structures from the pdb database that contain rna ( as of june 2007 ) . the base pairing patterns were ascertained using rnaview ( 18 ) . the junctionscanner program described in the previous section was used to parse the pdb and rnaview data . for each identified structural element the junctionscanner program generates two files : a file containing the extracted element in pdb format , and a text file describing all identified properties ( such as helix orientation , inter - helix angles , nomenclature , nucleotide sequences , residue indices ; the full list of properties is shown in the supplementary data ) . the junctionscanner algorithm was applied five times to each coordinate input file to avoid the unlikely chance that a helix was missed due to the random component inherent in the helix - fitting algorithm . the algorithm identified 258 kissing loop structures , 9357 internal loops and bulges , 2065 three - way junctions , 1091 four - way junctions , 462 five - way junctions , 70 six - way junctions , 23 seven - way junctions , one eight - way junction and one nine - way junction . five hundred and fifty - nine different pdb files contained structural elements meeting the minimum filter criteria described earlier . the counts for the number of identified junctions depend on how strict the parameters for the junction - detection algorithm are chosen . counts for the different junction types and junction scanner parameter sets are listed in table 2 . if a structural element is identified using several different filter parameter sets , only the version corresponding to the strictest parameter set is stored in the database , thus avoiding unnecessary redundancy . table 2.counts of identified rna junctions , internal loops , bulges and kissing loops the counts are given for three different filter parametersstructural elementmost strictmedium strictleast strictreduced redundancykissing loop9820625851internal loop / bulge31335635935718373-way junction731141220654314-way junction28049010912725-way junction109237462976-way junction4670177-way junction455561168-way junction01119-way junction0011the last column shows the counts for the reduced - redundancy least - restricted data set whose representative structures can be part of either the most - strict , medium - strict or least - strict data set . counts of identified rna junctions , internal loops , bulges and kissing loops the counts are given for three different filter parameters the last column shows the counts for the reduced - redundancy least - restricted data set whose representative structures can be part of either the most - strict , medium - strict or least - strict data set . all structural elements of the same type , consisting of the same sequences were clustered using single linkage clustering and a structural superposition cutoff of 3.0  drms ( c4 atom positions ) . for each cluster , one of its members this reduces the number of structural elements by a factor of about four ( compare table 2 ) . analyzing the structural variation within clusters , we found that in all but three cases the drms of the cluster members with respect to their cluster representative is smaller then 3.0  . the results of the junctionscanner applied to pdb coordinate files was entered into a mysql relational database . these data include the quantitative analysis , sequence , geometric conformation and citation for the identified structural elements as well as references to the parent structure from the pdb and the extracted coordinate data ( the full list of stored properties is given in the supplementary data ) . images of the secondary structure ( generated using rnaview ) and tertiary structure [ generated using raster3d ( 23 ) ] are available . the 3d coordinates of the structural element can be viewed interactively using the jmol java applet . the text output of the junctionscanner run is also provided and contains more detailed information such as the local coordinate systems of the fitted idealized helices . additional data is included in the form of structures obtained by molecular mechanics minimization , calculated using amber 8.0 ( 24 ) and the cornell force field ff99 ( 25 ) . analyzing the minimized structures , we found that 68% of them have an rmsd of < 1  compared to their respective original structure ; 31% have an rmsd between 1 and 2  , 0.7% exhibit an rmsd between 2 and 3  , 0.0003% show an rmsd between 3 and 5  . we plan to supplement the rnajunction database also with molecular dynamics simulation results in the future . the essential tables underlying the rnajunction database correspond to junctions , helices , strands , references and angles . in this way , helices and junctions , for example , are related through a many - to - one relationship , allowing in principle for junctions of arbitrarily large order . more detailed information about the design of the database tables is provided in the supplementary data . internal loops , bulges and kissing loops are internally considered a special case of  two - way junctions ; a flag indicates the different strand connectivity of a kissing loop compared to an internal loop . the aim of this database is 2-fold : first , it is an important resource for rna nanodesign , which requires a library of known rna structural elements for the development of novel rna structures ( 20,26 ) . second , we envision the database to be useful for analysis , in particular for understanding the principles of rna structure and folding . rnajunction ( http://rnajunction.abcc.ncifcrf.gov/ ) is publicly available and its data can be accessed through a user - friendly website . the rnajunction database contains detailed information about rna structural elements ( currently junctions , internal loops , bulges and kissing loops ) . using the web interface , the user can search for structural elements based on their nc - iubmb nomenclature ( 17 ) , type of junction , inter - helical angles , pdb identifier , the rnajunction identifier , sequence , experimental method , author of the corresponding published structure or parts of the text contained in the header records of the pdb structure . the results of a search are displayed by listing a summary of all matching structural elements . users may then click on a database identifier which points to a page providing more detailed information about a particular structural element . the database provides , in its detailed view , information about the sequences of a structural element , its nc - iubmb nomenclature , inter - helix angles . even more detailed information , such as the orientation of the connecting helices can be obtained by following the web links are provided for a literature citation as well as various related databases , such as the pdb ( 27 ) , scor ( 9,10 ) and mmdb ( 28 ) . using the jmol java applet ( http://www.jmol.org/ ) , the user can interactively display a 3d representation of a junction or kissing loop . as an example of the utility of the database , we show the result page of a kissing loop structure ( pdb i d 2bj2 , figure 3 ) . this structural element has been used for the design of an rna hexagonal ring ( 6 ) . the rnajunction database annotates this structure to possess an inter - helix angle of 122 ( see figure 3 bottom ) . thus , the added information makes it immediately clear that this structural element is a good candidate for designing a hexagonal structure requiring angles of 120. 
 figure 3.result page of an rna loop  loop interaction derived from pdb structure 2bj2 . the rnajunction database is freely available at http://rnajunction.abcc.ncifcrf.gov general queries can be performed using virtually any web browser ; displaying 3d structures with jmol requires the browser to be able to run java applets . the database is available for download ( structures , junctionscanner output , images ) upon request .
we developed a database called rnajunction that contains structure and sequence information for rna structural elements such as helical junctions , internal loops , bulges and loop  loop interactions . 
 our database provides a user - friendly way of searching structural elements by pdb code , structural classification , sequence , keyword or inter - helix angles . 
 in addition , the structural data was subjected to energy minimization . 
 this database is useful for analyzing rna structures as well as for designing novel rna structures on a nanoscale . 
 the database can be accessed at : http://rnajunction.abcc.ncifcrf.gov/