An efficient synthesis of new cap analogs containing 7-deazaguanosine moiety such as m(7)G[5']ppp[5'](7-deaza)G and m₂(7,3'O)G[5']ppp[5'](7-deaza)G is described. The biological substrate validation of these new cap analogs is evaluated with respect to its capping efficiency and in vitro T7 RNA polymerase transcription using standard cap m⁷G[5']ppp[5']G as a control. The capping efficiency and HPLC data reveal that these new analogs are not the substrate for T7 RNA polymerase or SP6 RNA polymerase. The present study highlights the importance of the presence of nitrogen atom at N7-position of the guanosine moiety for the polymerase recognition.