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The calculation takes only inter-molecular base pairs into account, for the general case use RNAcofold. The use of alignments allows to focus on binding sites that are evolutionary conserved. Note, that the two input alignments need to have equal number of sequences and the same order, i.e. the 1st sequence in file1 will be hybridized to the 1st in file2 etc. The computed binding sites, energies, and structures are written to stdout, one structure per line. Each line consist of: The structure in dot bracket format with a "&" separating the two strands. The range of the structure in the two sequences in the format "from,to : from,to"; the energy of duplex structure in kcal/mol. The format is especially useful for computing the hybrid structure between a small probe sequence and a long target sequence. -h, --help Print help and exit --detailed-help Print help, including all details and hidden options, and exit -V, --version Print version and exit Below are command line options which alter the general behavior of this program -e, --deltaEnergy=range Compute suboptimal structures with energy in a certain range of the optimum (kcal/mol). Default is calculation of mfe structure only. -s, --sorted print output sorted by free energy (default=off) -T, --temp=DOUBLE Rescale energy parameters to a temperature of temp C. Default is 37C. -4, --noTetra Do not include special tabulated stabilizing energies for tri-, tetra- and hexaloop hairpins. Mostly for testing. (default=off) -d, --dangles=INT How to treat "dangling end" energies for bases adjacent to helices in free ends and multi-loops (default=`2') With -d1 only unpaired bases can participate in at most one dangling end, this is the default for mfe folding but unsupported for the partition function folding. With -d2 this check is ignored, dangling energies will be added for the bases adjacent to a helix on both sides in any case; this is the default for partition function folding (-p). -d0 ignores dangling ends altogether (mostly for debugging). With -d3 mfe folding will allow coaxial stacking of adjacent helices in multi-loops. At the moment the implementation will not allow coaxial stacking of the two interior pairs in a loop of degree 3 and works only for mfe folding. Note that by default (as well as with -d1 and -d3) pf and mfe folding treat dangling ends differently. Use -d2 in addition to -p to ensure that both algorithms use the same energy model. --noLP Produce structures without lonely pairs (helices of length 1). (default=off) For partition function folding this only disallows pairs that can only occur isolated. Other pairs may still occasionally occur as helices of length 1. --noGU Do not allow GU pairs (default=off) --noClosingGU Do not allow GU pairs at the end of helices (default=off) --noconv Do not automatically substitude nucleotide "T" with "U" (default=off) --nsp=STRING Allow other pairs in addition to the usual AU,GC,and GU pairs. Its argument is a comma separated list of additionally allowed pairs. If the first character is a "-" then AB will imply that AB and BA are allowed pairs. e.g. RNAfold -nsp -GA will allow GA and AG pairs. Nonstandard pairs are given 0 stacking energy. -P, --paramFile=paramfile Read energy parameters from paramfile, instead of using the default parameter set. A sample parameter file should accompany your distribution. See the RNAlib documentation for details on the file format. See also: RNAduplex(l) RNAcofold(l) RNAfold(l) Written by: Ivo L Hofacker Report bugs: If in doubt our program is right, nature is at fault. Comments should be sent to rna@tbi.univie.ac.at M@"O@3O@N@`O@O@%s %3d,%-3d : %3d,%-3d (%5.2f) unequal number of seqs in alignmentscan't open %s No input filesB?-d%d -T %f B@ INF -INf DEF%6dstackstack_enthalpiesbulgebulge_enthalpiesmismatch_exteriormismatch_exterior_enthalpiesmismatch_hairpinmismatch_hairpin_enthalpiesmismatch_interiormismatch_interior_enthalpiesmismatch_interior_1nmismatch_interior_23mismatch_multimismatch_multi_enthalpiesint11int11_enthalpiesint21int21_enthalpiesint22int22_enthalpiesdangle5dangle5_enthalpiesdangle3dangle3_enthalpiesML_paramsNINIOTriloopsTetraloopsHexaloopsMiscEND The answer is: 42 NPCGGCUGAUUA @can't open file %s # %s /* %2s..%2s */ /* %2s.%c..%2s */ /* %2s.%c%c..%2s */ %6d %6d %6d %6d %6d %6d %.8s %6d %6d %.6s %6d %6d %.5s %6d %6d @ACGU/**/can't extrapolate first valueNST%15s%n rd_1dim: %s File %s is inproper. # %255s%6s %d %d%5s %d %d%8s %d %dmismatch_interior_1n_enthalpiesmismatch_interior_23_enthalpies## RNAfold parameter file v2.0 /* CG GC GU UG AU UA @ */ /* @ A C G U */ /* F = cu*n_unpaired + cc + ci*loop_degree (+TermAU) */ /* cu cu_dH cc cc_dH ci ci_dH */ /* Ninio = MIN(max, m*|n1-n2| */ /* m m_dH max */ %6d %6d %6d /* all parameters are pairs of 'energy enthalpy' */ /* DuplexInit TerminalAU LXC */ %6d %6d %6d %6d %3.6f %6d unexpected end of file in get_array1unclosed comment in parameter file read_parameter_file: can't open file %s using default parameters instead. ## RNAfold parameter file v2.0Missing header line in file. May be this file has not v2.0 format. Use INTERRUPT-key to stop. read_epars: Unknown field identifier in `%s' WARNING: stacking energies not symmetric WARNING: stacking enthalpies not symmetric WARNING: int11 energies not symmetric (%d,%d,%d,%d) (%d vs. %d) WARNING: int11 enthalpies not symmetric WARNING: int22 energies not symmetric WARNING: int22 enthalpies not symmetric: %d %d %d %d %d %d $-6?HQZclu~ɰհ)5AMYeq}p@X`x6?HT!*[Vn{%sInput structure constraints using the following notation:< : base i is paired with a base j : base i is paired with a base j>imatching brackets ( ): base i pairs base jInput string (upper or lower case)SPACE allocation failure -> EINVALSPACE allocation failure -> no memoryunbalanced brackets in constraintunbalanced brackets in constraintsunbalanced brackets in constraint stringunbalanced brackets in make_pair_tablepack_structure: illegal charcter in structurexrealloc allocation failure -> EINVALxrealloc allocation failure -> no memoryconstraints of type '|' not allowedconstraints of type '<' or '>' not allowedconstraints of type '(' or ')' not allowedconstraints of type 'x' not allowedunrecognized character in constraint structure333333?xxxxx,OxxxxxABCDEFGHIJKLMNOPQRSTUVWXYZabcdefghijklmnopqrstuvwxyzenergy_of_ml_pt: i is not 5' base of a closing pair!What energy_set are YOU using??initialize_fold: argument must be greater 0WARNING: bases %d and %d (%c%c) can't pair! Interior loop (%3d,%3d) %c%c; (%3d,%3d) %c%c: %5d Hairpin loop (%3d,%3d) %c%c : %5d Multi loop (%3d,%3d) %c%c : %5d External loop : %5d energy_of_struct: string and structure have unequal lengthi is unpaired in loop_energy() could not enforce all constraints >@@ư>backtrack failed in f5backtrack failed in fMLbacktracking failed in repeatfffffq@%zr?$@x[f+Pz?IHbQ??t@$@out of memory%s %6d %6d %s %6.2f Error while backtracking!%d,%dOops, no solution in repeat! repeat: Warning: %d %d can't pair energy range too high, limiting to reasonable valueY@backtrack failed in f5 (or fc)backtrack failed in fc[] 5' of cutinit_cofold: argument must be greater 0@@Y?{Gz?Abacktrack failed in fold duplexunequal number of sequences in alibacktrack() unequal number of sequences in aliduplexfold() backtrack failed in aliduplex%d %d %d uneqal seqence lengthsoJ?%f %f %f %f %f %falloc_sequence_arrays: no sequences in the alignment!energy_of_alistruct(): no sequences in alignment!Empty CLUSTAL file CLUSTALSTOCKHOLM//%99s %sThis doesn't look like a CLUSTAL/STOCKHOLM file, sorry Sorry, your file is fucked up (inconsitent seq-names) Too many sequences in CLUSTAL/STOCKHOLM fileNo sequences found in CLUSTAL/STOCKHOLM file Sorry, your file is fucked up. 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W>?}>4f??@0@a>%#*@@?@-n?P>Z/ @~@0@0@P>>R?\s>j?a>Z/ @R?7>T?F>%#*@~@\s>T?D6>?@0@j?F>?>GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-46)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-46)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-48)GCC: (GNU) 4.1.2 20080704 (Red Hat 4.1.2-46),PE@5, b@, Ph@,L&i@$=,N@N,``@ ,pA>,AcS,9p1B,k 4B),RPBCH,4C},n\C,c0CT RNAaliduplex_cmdline_parser_initfRNAaliduplex_cmdline_parser_params_initRNAaliduplex_cmdline_parser_required RNAaliduplex_cmdline_parser_dump_ RNAaliduplex_cmdline_parser_params_create RNAaliduplex_cmdline_parser_free; 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"vKpg!gseYgDy##YYu=ejׁu8=>agggLp(~[quu+g&[G\p:hIueh/s!gB?;\*j]0uZHvuZV>[  Kםy*\ tv.TX,JK#[-KIuWu+3؅f^6##T ./../H/usr/include/usr/include/bits/usr/lib/gcc/x86_64-redhat-linux/4.1.2/includeparams.cdata_structures.hstdio.hlibio.htypes.hstddef.henergy_par.hfold_vars.h pA!h{"hSyL)0,"r=D*dh$K@=D3wqL -wt F/g3#:!gP$:!gP$:!gBus/M>Dkus/5r/]SB;$ TAX/;vL,~X~M?JKA+Koz/-K/?K/s-c%S,w2A#wI?UMM,I?MM,~I?MM, M}@3S w.yLuW=KsgK@$K,I@0q *wt w 6,g3#:!gM$:!gM$:!g?us/JL@kus/2r0MY?;$ TAX/;vL)f~X~M?GKA+Kow/*K/<K,s-c%W%w2A#wI?UMM)I?MM)I?MM) M}@ ut vrLI3.6m+ ttL -wt D*g4=I=I=9G1]us/-q7WB;Ku/uwtTX.;vL+f~SpI/--J-=.urD=.Y=b8(23L(wI?UMM,I?MM,I?MM, YYZ%"؟s={3S.&$#&jzxvPztPzrvsusu3;u<;u<;u.*rvrvMj?AX-ueKffjMy%Ks()vr#1s=s0;Mt6=$=yatD-ueKǝtS-ueKƝts-ueK ggh!"؟s=   ./../H/usr/lib/gcc/x86_64-redhat-linux/4.1.2/include/usr/include/usr/include/bitssubopt.cpair_mat.hloop_energies.hdata_structures.hlist.hstddef.hstdio.hlibio.htypes.hfold_vars.hfold.henergy_par.h Ag;Y~ <yYK%/Y;=:>r zXyL:Zvx%SۃG f;K0t"KK>K:T J!h~f ==u&* rt6-uh|oJI7p Y<7XI'tY'Y,5%2fZqwrvcwHv=y)g $wfsuyyt/t)ZHY=Y=7YY9iqyXy.Jm xT<x  /~f-L#3q?#y[<yiU=YZuI=\V؜>dX"<rY:vyXyt/f"A-=s=Y=;gsu*['==i9Mq?~#[,+%y K;Y=- XyiiHhu=Y*=I=Yy>yZ(ByJyf  y(y  PhL_==*;Y=1*bz$"G`9 UxxzWl ;Z:h$1;Y0us=|t{.,{0-u-/K<{1-s-u Xvt t/KWgjY=;=}X:>gQeKI=It̥yt j t./ɯI=0ZH9~Xd~.J~J~t+vYs <su&KU{ {%=e  ggg}'vy X<{1-s-LI00~60 1P@=I=Yv[>N}1gt0 9)BN|X|lf|t/fgmQ/><|.|K?Y|X|t/f"6.z  f}t/fg@=su/d[zPz<}grhqJ5LqM}f}t/f}<X}/%-=I=Zsux|6X|<{J{*u妤 =l:(wu;(x"&6$I0/-uhʟu fw.|~XyP/7Z/{ft{<{ ˅:hX tvt tvJ Jvt.=I=Y=I=YKtJ{fJ{XfTV` J>nb]Jw;*-:P+H'K4U?I=[FYI=[=I=[vz5 ?X(/-Pz5Oqz2 #gt$tJ:?z.K`s=I=Z}<K}I~Xfzt/fyFD=s=I=Y$~J)UByQysY1zXzXPgt\f/&%<:@/{'g?Yf"uB6r>z)X{tgIYs=I=Y;zjT?<'}X}(%=I=Yx<hg|%v}(~f%fILrY4KǃFtY;=z2=W~1Yf"@{~.=I=Y~<=I=ZJ<zdhg|%vn4Yf"JzXztg=sg /usr/include/usr/include/bits/usr/lib/gcc/x86_64-redhat-linux/4.1.2/includelist.clist.hstdio.hlibio.htypes.hstddef.h p1B:HLKLL;=IMuK LIw8vZ fuJ =;Z=&=;X<<<< Ltf"FzztXY-[t==<gX<g<_yJh=f/ְ!X~ u#=pm=;Gf M"gTMGM/ ./../H/usr/include/usr/include/bits/usr/lib/gcc/x86_64-redhat-linux/4.1.2/includecofold.cloop_energies.hpair_mat.hdata_structures.hstdio.hlibio.htypes.hstddef.henergy_par.hfold_vars.hfold.h  4B(0rKs=K",LVGKgK^]nXzytdt#X]XX^yr zXyrpCyW󁃠irdgh<hf{U=ɘ 2~;/ #woXJvr< `ztNkJt~o;K@~<.~<~X/1Y}.}KPY3!;KuN}.}YMg?}fgi<<~hu8vs9z&zuuiK 3}ttk}<}tk<t}}}<}<p}J}t}<}t2uK}<}}J}Y;Y=%Ƅg-W׫$h٣n*RiJi} ~thx8vrx>wu@~4u #YtzXK;K/&@~f<p?{f{t/t{KMgt{KPgt'{b #gt$?hu}}u({` #gtם/{h<{{z</gY}qwR}}u(|.srg|>*}䃁;Y=*}pr~gg/sh|f}4f$A3s~<~}XxYtx|x<}f}<(}Xixf}3! 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