oligoribonucleotides were synthesized on an mm12 synthesizer ( bioautomation corp . , irving , tx ) on a 50  nmol scale with 2otbdmsphosphoramidites ( thermo fisher ) , using a 1000   unylinker support ( chemgenes , wilmington , ma ) .
coupling time was 290  s for regular 2otbdms phosphoramidites , 2180  s for the dnb phosphoramidite 5 , and 3240  s for 2opropargyl rc phosphoramidite .
isolated 5dnbprotected oligonucleotides were dried in a speedvac and redissolved in 200  l rnasefree water prior to irradiation for 10  min at 365  nm in a vilber lourmat biolink blx crosslinker ( 58  w ) .
final rphplc purification yielded 5phosphate oligoribonucleotides ready for the introduction of functional groups by click chemistry ( e.g. , psoralen , cy5 ) , and for enzymatic ligation experiments with t4  rna ligase  2 ( m0239 , new england biolabs ) ( see the supporting information for details ) .
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 abstractlong structured rnas are useful biochemical and biological tools . 
 they are usually prepared enzymatically , but this precludes their sitespecific modification with functional groups for chemical biology studies . 
 one solution is to perform solidphase synthesis of multiple rnas loaded with 5terminal phosphate groups , so that rnas can be concatenated using template ligation reactions . 
 however , there are currently no readily available reagents suitable for the incorporation of the phosphate group into long rnas by solidphase synthesis . here 
 we describe an easytoprepare phosphoramidite reagent suitable for the chemical introduction of 5terminal phosphate groups into long rnas . 
 the phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide byproducts . 
 the phosphate is unmasked quantitatively by brief uv irradiation . 
 we demonstrate the value of this reagent in the preparation of a library of long structured rnas that are sitespecifically modified with functional groups . 
Experimental Section
Conflict of interest
Supporting information